A method for detecting the hemagglutination titer of an influenza virus antigen sample

Abstract

The present invention discloses a method for detecting the hemagglutination titer of an influenza virus antigen sample, comprising the following steps: S1. Lysis treatment: adding a lysing agent to the influenza virus antigen sample and the influenza virus antigen hemagglutination titer reference product respectively for lysis to obtain a lysed influenza virus antigen sample and a lysed influenza virus antigen hemagglutination titer reference product; the influenza virus antigen sample is an intermediate produced in each process during the production of the monovalent bulk of the influenza virus split vaccine; S2. Hemagglutination titer detection: detecting the hemagglutination titer of the lysed influenza virus antigen sample and the hemagglutination titer of the lysed influenza virus antigen hemagglutination titer reference product respectively; S3. Calculating the standard value of the hemagglutination titer of the influenza virus antigen sample. The detection method of the present application has good precision, good repeatability and good accuracy, and can be used for the quality control evaluation of intermediates in each process during the production of the monovalent bulk of the influenza virus split vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to a detection method for biological agents, in particular to a detection method for the hemagglutination titer of an influenza virus antigen sample. Background Art

[0002] According to statistics, about 5% - 15% of the global population is infected with influenza virus every year, resulting in 3 - 5 million severe cases and 500,000 deaths. Influenza vaccination is the most effective preventive measure against influenza virus infection. Hemagglutination titer, as a quality evaluation index for virus allantoic fluid harvest, has the advantages of simple method and good flexibility. Currently, the conventional hemagglutination method is often used to detect the hemagglutination titer of influenza virus allantoic fluid harvest. The principle of the conventional hemagglutination method is that influenza virus, parainfluenza virus, etc. selectively agglutinate the red blood cells of individual animals. The materials required for measuring the hemagglutination titer by the conventional hemagglutination method are: 1v% chicken blood red blood cell suspension, 0.9wt% sodium chloride solution, and a microtiter plate (U-shaped). The steps for measuring the hemagglutination titer by the conventional hemagglutination method are as follows: Add 50 μL of 0.9wt% sodium chloride solution to each well of the microtiter plate. Add the sample virus solution to the first well, mix well by repeatedly pipetting and sucking, and aspirate 50 μL and transfer it to the second well; and so on, perform equal-volume serial dilution until the 12th well, aspirate 50 μL from the 12th well and discard it, and dilute the virus in this way; set up two other wells, add 50 μL of 0.9wt% sodium chloride solution to each well as a negative control, add 50 μL of 1v% chicken blood red blood cell suspension to each well, mix well and let stand for 30 min, and then determine the test results of the hemagglutination test.

[0003] The production process of the monovalent bulk of influenza virus split vaccine mainly includes steps such as virus inoculation and culture, virus harvest, allantoic fluid harvest merging, virus inactivation, ultrafiltration concentration, purification, virus lysis, post-lysis purification, and sterile filtration. How to achieve the quality control and evaluation of the intermediates in each process of the production process of the monovalent bulk of influenza virus split vaccine is very important.

[0004] However, the applicant found in the research process that the conventional hemagglutination method has the following deficiencies when used to detect the hemagglutination titer of the intermediates in each process of the production process of the monovalent bulk of influenza virus split vaccine: 1) Factors such as the quality, concentration, and storage time of the chicken blood cells used for detection have an impact on the test results of the conventional hemagglutination method; 2) Since the physical state of the virus antigen in the intermediates such as virus concentrate, purified solution, lysate, and bulk is quite different from that of the virus allantoic fluid harvest, the sample state has a great impact on the test results of the conventional hemagglutination method. Therefore, it is impossible to accurately and objectively control and evaluate the quality of the intermediates in each process of the production process of the monovalent bulk of influenza virus split vaccine using the conventional hemagglutination method. Summary of the Invention

[0005] In order to solve the problem that the conventional hemagglutination method cannot accurately and objectively evaluate the quality control of intermediates in each process of the production of monovalent bulk influenza virus split vaccine, the present application provides a method for detecting the hemagglutination titer of influenza virus antigen samples.

[0006] In a first aspect, the present application provides a method for detecting the hemagglutination titer of influenza virus antigen samples, which is implemented by the following technical solutions:

[0007] A method for detecting the hemagglutination titer of influenza virus antigen samples includes the following steps:

[0008] S1. Lysis treatment: Add a lysing agent to the influenza virus antigen sample and the influenza virus antigen hemagglutination titer reference product respectively for lysis to obtain a lysed influenza virus antigen sample and a lysed influenza virus antigen hemagglutination titer reference product; the influenza virus antigen sample is an intermediate produced in each process of the production of monovalent bulk influenza virus split vaccine.

[0009] S2. Hemagglutination titer detection: Detect the hemagglutination titer of the lysed influenza virus antigen sample and the hemagglutination titer of the lysed influenza virus antigen hemagglutination titer reference product respectively to obtain the hemagglutination titer detection value of the influenza virus antigen sample and the hemagglutination titer detection value of the influenza virus antigen hemagglutination titer reference product.

[0010] S3. Calculate the hemagglutination titer standard value of the influenza virus antigen sample: Use the ratio between the hemagglutination titer standard value of the influenza virus antigen hemagglutination titer reference product and the hemagglutination titer detection value of the influenza virus antigen hemagglutination titer reference product as a correction coefficient to correct the hemagglutination titer detection value of the influenza virus antigen sample to obtain the hemagglutination titer standard value of the influenza virus antigen sample.

[0011] By adopting the above technical solutions, the present application lyses the influenza virus antigen sample, thereby achieving the consistency of the physical properties of the intermediates produced in each process of the production of monovalent bulk influenza virus split vaccine, effectively avoiding the influence of the state of the intermediates produced in each process of the production of monovalent bulk influenza virus split vaccine on the detection results of the conventional hemagglutination method, improving the detection accuracy, and more precisely controlling and evaluating the quality of the intermediates in each process of the production of monovalent bulk influenza virus split vaccine. At the same time, the influenza virus antigen sample and the influenza virus antigen hemagglutination titer reference product are lysed synchronously, effectively reducing the influence of the operation process on the detection results. In addition, in the detection process of the hemagglutination titer of the present application, the influenza virus antigen hemagglutination titer reference product is used to effectively eliminate the influence of factors such as the quality, concentration, and storage time of chicken red blood cells on the detection results, improving the stability of the detection results.

[0012] Preferably, the influenza virus antigen sample includes any one of an influenza virus harvest fluid, an influenza virus deglycosylated and purified fluid, an influenza virus lysate, an influenza virus post-lysis purified fluid, or a monovalent bulk influenza virus split vaccine.

[0013] In this application, the influenza virus harvest fluid is a sample obtained after the ultrafiltration and concentration process in the production process of the monovalent stock solution of the influenza virus split vaccine. Specifically: Dilute the working seed (influenza virus) by inoculating the allantoic cavity of chicken embryos and culture for 48 - 72 hours; After cultivation, select live chicken embryos, harvest the allantois into a container and perform inspection on the allantoic fluid harvest in each harvest container, combine the qualified allantoic fluids to obtain a monovalent influenza virus combined fluid; Add formaldehyde with a concentration not higher than 200 μg / mL to the monovalent influenza virus combined fluid for virus inactivation to obtain an inactivated monovalent influenza virus combined fluid; Centrifuge the inactivated monovalent influenza virus combined fluid and then concentrate it by the ultrafiltration method to obtain the influenza virus harvest fluid.

[0014] In this application, the influenza virus deglycosylated and purified fluid is a sample obtained after the purification process in the production process of the monovalent stock solution of the influenza virus split vaccine. Specifically: Purify the influenza virus harvest fluid by sucrose density gradient centrifugation to obtain the influenza virus deglycosylated and purified fluid.

[0015] In this application, the influenza virus split fluid is a sample obtained after the virus splitting process in the production process of the monovalent stock solution of the influenza virus split vaccine. Specifically: Add a splitting agent to the influenza virus deglycosylated and purified fluid for virus splitting to obtain the influenza virus split fluid.

[0016] In this application, the influenza virus post - split purified fluid is a sample obtained after the post - split purification process in the production process of the monovalent stock solution of the influenza virus split vaccine. Specifically: Repurify the influenza virus split fluid by methods such as column chromatography or sucrose density gradient centrifugation to obtain the influenza virus post - split purified fluid.

[0017] In this application, the monovalent stock solution of the influenza virus split vaccine is a sample obtained after the sterilization and filtration process in the production process of the monovalent stock solution of the influenza virus split vaccine. Specifically: Sterilize the influenza virus post - split purified fluid to obtain the monovalent stock solution of the influenza virus split vaccine.

[0018] The method for detecting the hemagglutination titer of the influenza virus antigen sample provided in this application can accurately analyze the hemagglutination titers of the influenza virus harvest fluid, the influenza virus deglycosylated and purified fluid, the influenza virus split fluid, the influenza virus post - split purified fluid, and the monovalent stock solution of the influenza virus split vaccine.

[0019] Preferably, the monovalent stock solution of the influenza virus split vaccine includes any one of the monovalent stock solution of H1N1 influenza virus, the monovalent stock solution of H3N2 influenza virus, the monovalent stock solution of BV influenza virus, or the monovalent stock solution of BY influenza virus.

[0020] In this application, the influenza virus harvest fluid includes any one of the H1N1 influenza virus harvest fluid, the H3N2 influenza virus harvest fluid, the BV influenza virus harvest fluid, or the BY influenza virus harvest fluid.

[0021] In this application, the influenza virus deglycosylated and purified fluid includes any one of the H1N1 influenza virus deglycosylated and purified fluid, the H3N2 influenza virus deglycosylated and purified fluid, the BV influenza virus deglycosylated and purified fluid, or the BY influenza virus deglycosylated and purified fluid.

[0022] In this application, the influenza virus lysate includes any one of the H1N1 influenza virus lysate, the H3N2 influenza virus lysate, the BV influenza virus lysate, or the BY influenza virus lysate.

[0023] In this application, the influenza virus purified lysate includes any one of the H1N1 influenza virus purified lysate, the H3N2 influenza virus purified lysate, the BV influenza virus purified lysate, or the BY influenza virus purified lysate.

[0024] The detection method for the hemagglutination titer of the influenza virus antigen sample provided in this application has a better detection effect on various influenza viruses, the stability of the detection result is high, and the detection result can be used to evaluate the quality control of intermediates in each process during the production of the H1N1 influenza virus monovalent stock solution, the H3N2 influenza virus monovalent stock solution, the BV influenza virus monovalent stock solution, or the BY influenza virus monovalent stock solution.

[0025] Preferably, in the S1 step, the hemagglutinin content of the influenza virus antigen sample before lysis is 10 - 50 μg / mL; the influenza virus hemagglutination titer reference product needs to be reconstituted with 0.5 mL of sterile injection water before adding the lysing agent to obtain the influenza virus hemagglutination titer reference product solution.

[0026] In this application, the hemagglutinin content of the influenza virus harvest fluid before lysis can be 10 - 50 μg / mL; however, the influenza virus deglycosylated and purified fluid, the influenza virus lysate, the influenza virus purified lysate, and the influenza virus lysate vaccine monovalent stock solution need to be diluted with PBS buffer to a hemagglutinin content of 10 - 50 μg / mL. The diluted liquids of the influenza virus deglycosylated and purified fluid, the influenza virus lysate, the influenza virus purified lysate, and the influenza virus lysate vaccine monovalent stock solution are respectively denoted as the diluted influenza virus deglycosylated and purified fluid, the diluted influenza virus lysate, the diluted influenza virus purified lysate, and the diluted influenza virus lysate vaccine monovalent stock solution.

[0027] Preferably, the lysing agent is selected from any one of Triton X - 100, polysorbate 80, Triton N101, or Detergent.

[0028] More preferably, in the step S1, the lysing agent is 10 wt% Detergent.

[0029] Preferably, in the step S1, the volume ratio of the influenza virus antigen sample to the lysing agent before lysis and the volume ratio of the influenza virus antigen hemagglutination titer reference product solution to the lysing agent are both (8-10):1.

[0030] More preferably, in the step S1, the volume ratio of the influenza virus antigen sample to the lysing agent before lysis and the volume ratio of the influenza virus antigen hemagglutination titer reference product solution to the lysing agent are both 9:1.

[0031] In the present application, the volume ratio of the influenza virus harvest fluid to the lysing agent is 9:1, and the volume ratios of the influenza virus deglycosylated purification fluid dilution, the influenza virus lysate dilution, the influenza virus post-lysis purification fluid dilution, and the influenza virus split vaccine monovalent stock solution dilution to the lysing agent are all 9:1.

[0032] Preferably, in the step S1, the time for the lysis treatment is 20-40 min.

[0033] More preferably, in the step S1, the time for the lysis treatment is 30 min.

[0034] Preferably, in the step S2, the specific steps for the hemagglutination titer detection include:

[0035] S21. Marking the hemagglutination plate;

[0036] S22. Serial dilution: Add 50 μL of the lysed influenza virus antigen sample and the lysed influenza virus antigen hemagglutination titer reference product to the first well of the hemagglutination plate respectively. Take out 50 μL from the first well and add it to the second well, pipette and mix well, and perform a 2-fold serial dilution in sequence until a negative result of red blood cell agglutination appears. Discard 50 μL after mixing the last well;

[0037] S23. Adding chicken red blood cell suspension;

[0038] S24. Excluding non-specific agglutination of red blood cells by the negative control;

[0039] S25. Incubation;

[0040] S26. Calculating the hemagglutination titer detection value: Observe the red blood cell agglutination result, take the previous positive before the negative result appears as the determination end point, and calculate the hemagglutination titer detection value of the influenza virus antigen sample and the hemagglutination titer detection value of the influenza virus antigen hemagglutination titer reference product respectively.

[0041] In the present application, the red blood cell agglutination results are specifically shown in Table 1.

[0042] Table 1 Results of Red Blood Cell Agglutination

[0043]

[0044] As can be seen from Table 1, in this application, a negative result means that the red blood cells do not agglutinate 100%, that is, the phenomenon corresponding to "-". The previous positive result before the negative result refers to the phenomenon before the appearance of "-", that is, it may be one of the phenomena corresponding to "++++", "+++", "++" or "+".

[0045] Table 2 Calculation Method of Hemagglutination Titer

[0046] Result Calculation method of hemagglutination titer ++++ 1: (Dilution factor of the previous positive before the negative result × 3 / 2) +++ 1: (Dilution factor of the previous positive before the negative result × 5 / 4) ++ 1: Dilution factor of the previous positive before the negative result + 1: (Dilution factor of the previous positive before the negative result × 7 / 8)

[0047] Preferably, in step S3, the calculation formula for the standard value of the hemagglutination titer of the influenza virus antigen sample is as follows:

[0048]

[0049] where T is the standard value of the hemagglutination titer of the influenza virus antigen sample;

[0050] T t is the hemagglutination titer detection value of the influenza virus antigen sample;

[0051] T s is the hemagglutination titer detection value of the influenza virus hemagglutination titer reference product;

[0052] T0 is the standard value of the hemagglutination titer of the influenza virus hemagglutination titer reference product.

[0053] In this application, the H1N1 influenza virus harvest fluid, H1N1 influenza virus deglycosylated and purified fluid, H1N1 influenza virus lysate, H1N1 influenza virus lysed and purified fluid, or H1N1 influenza virus lysate vaccine monovalent stock solution use the same influenza virus hemagglutination titer reference product, that is, the H1N1 influenza virus hemagglutination titer reference product; the standard value of the hemagglutination titer of the H1N1 influenza virus hemagglutination titer reference product is 1:515, that is, T0 = 1:515.

[0054] The H3N2 influenza virus harvest fluid, H3N2 influenza virus deglycosylated and purified fluid, H3N2 influenza virus lysate, H3N2 influenza virus lysed and purified fluid, or H3N2 influenza virus lysate vaccine monovalent stock solution use the same influenza virus hemagglutination titer reference product, that is, the H3N2 influenza virus hemagglutination titer reference product; the standard value of the hemagglutination titer of the H3N2 influenza virus hemagglutination titer reference product is 1:503, that is, T0 = 1:503.

[0055] The harvest fluid of influenza virus type BV, the deglycosylated and purified fluid of influenza virus type BV, the lysate of influenza virus type BV, the purified fluid after lysis of influenza virus type BV, or the monovalent stock solution of influenza virus type BV lysate vaccine uses the same reference product for the hemagglutination titer of influenza virus antigen, that is, the reference product for the hemagglutination titer of influenza virus type BV; the standard value of the hemagglutination titer of the reference product for the hemagglutination titer of influenza virus type BV is 1:1492, that is, T0 = 1:1492.

[0056] The harvest fluid of influenza virus type BY, the deglycosylated and purified fluid of influenza virus type BY, the lysate of influenza virus type BY, the purified fluid after lysis of influenza virus type BY, or the monovalent stock solution of influenza virus type BY lysate vaccine uses the same reference product for the hemagglutination titer of influenza virus antigen, that is, the reference product for the hemagglutination titer of influenza virus type BY; the standard value of the hemagglutination titer of the reference product for the hemagglutination titer of influenza virus type BY is 1:786, that is, T0 = 1:786.

[0057] In summary, the present application has the following beneficial effects:

[0058] 1. By lysing the influenza virus antigen sample, the present application achieves the consistency of the physical properties of the intermediates generated in each process during the production of the monovalent stock solution of influenza virus lysate vaccine, effectively avoiding the influence of the state of the intermediates generated in each process during the production of the monovalent stock solution of influenza virus lysate vaccine on the detection results of the conventional hemagglutination method, improving the detection accuracy, and more precisely controlling and evaluating the quality of the intermediates in each process during the production of the monovalent stock solution of influenza virus lysate vaccine.

[0059] 2. The present application lyses the influenza virus antigen sample and the reference product for the hemagglutination titer of influenza virus antigen synchronously, effectively eliminating the influence of the operation process on the detection results.

[0060] 3. During the detection process of the hemagglutination titer of the present application, the reference product for the hemagglutination titer of influenza virus antigen effectively eliminates the influence of factors such as the quality, concentration, and storage time of chicken red blood cells on the detection results, improving the stability of the detection results.

[0061] 4. The detection method for the hemagglutination titer of the influenza virus antigen sample has a wide applicability and high stability of the detection results. Specific Embodiments

[0062] The following further elaborates on the present application with reference to examples.

[0063] The harvest fluid of influenza virus, the deglycosylated and purified fluid of influenza virus, the lysate of influenza virus, the purified fluid after lysis of influenza virus, or the monovalent stock solution of influenza virus lysate vaccine in the present application all originate from the sampling of the intermediates in each process during the production of the monovalent stock solution of influenza virus lysate vaccine in our company, and three batches are sampled for each.

[0064] Among them, the three batches of samples of the H1N1 influenza virus harvest fluid are respectively recorded as Shou A1-1, Shou A1-2, and Shou A1-3;

[0065] The three batches of samples of the H1N1 influenza virus deglycosylated and purified fluid are respectively recorded as Tuo A1-1, Tuo A1-2, and Tuo A1-3;

[0066] The three batches of samples of the H1N1 influenza virus lysate are respectively recorded as Lie A1-1, Lie A1-2, and Lie A1-3;

[0067] The three batches of samples of the H1N1 influenza virus purified lysate are respectively recorded as Chun A1-1, Chun A1-2, and Chun A1-3;

[0068] The three batches of samples of the monovalent bulk of the H1N1 influenza virus split vaccine are respectively recorded as A1-1, A1-2, and A1-3;

[0069] The three batches of samples of the H3N2 influenza virus harvest fluid are respectively recorded as Shou A3-1, Shou A3-2, and Shou A3-3;

[0070] The three batches of samples of the H3N2 influenza virus deglycosylated and purified fluid are respectively recorded as Tuo A3-1, Tuo A3-2, and Tuo A3-3;

[0071] The three batches of samples of the H3N2 influenza virus lysate are respectively recorded as Lie A3-1, Lie A3-2, and Lie A3-3;

[0072] The three batches of samples of the H3N2 influenza virus purified lysate are respectively recorded as Chun A3-1, Chun A3-2, and Chun A3-3;

[0073] The three batches of samples of the monovalent bulk of the H3N2 influenza virus split vaccine are respectively recorded as A3-1, A3-2, and A3-3;

[0074] The three batches of samples of the BV influenza virus harvest fluid are respectively recorded as Shou BV-1, Shou BV-2, and Shou BV-3;

[0075] The three batches of samples of the BV influenza virus deglycosylated and purified fluid are respectively recorded as Tuo BV-1, Tuo BV-2, and Tuo BV-3;

[0076] The three batches of samples of the BV influenza virus lysate are respectively recorded as Lie BV-1, Lie BV-2, and Lie BV-3;

[0077] The three batches of samples of the BV influenza virus purified lysate are respectively recorded as Chun BV-1, Chun BV-2, and Chun BV-3;

[0078] The three batches of samples of the monovalent bulk of the BV influenza virus split vaccine are respectively recorded as BV-1, BV-2, and BV-3;

[0079] The three batches of samples of the BY influenza virus harvest fluid are respectively denoted as Harvest BY-1, Harvest BY-2, and Harvest BY-3;

[0080] The three batches of samples of the deglycosylated and purified fluid of the BY influenza virus are respectively denoted as Deglycosylate BY-1, Deglycosylate BY-2, and Deglycosylate BY-3;

[0081] The three batches of samples of the lysate of the BY influenza virus are respectively denoted as Lyse BY-1, Lyse BY-2, and Lyse BY-3;

[0082] The three batches of samples of the purified fluid after lysis of the BY influenza virus are respectively denoted as Purify BY-1, Purify BY-2, and Purify BY-3;

[0083] The three batches of samples of the monovalent bulk of the inactivated BY influenza virus vaccine are respectively denoted as BY-1, BY-2, and BY-3.

[0084] The lysing agent used in this application can be obtained commercially. Among them, Detergent is purchased from EMD Milipore.

[0085] The reference standards for the hemagglutination titer of the influenza virus antigen used in this application are all self-made.

[0086] Preparation Example 1 provides a reference standard for the hemagglutination titer of the H1N1 influenza virus. The preparation steps are as follows:

[0087] S1. Add 30 mL of water to a container. After heating and boiling, add 10 g of maltose weighed, stir until completely dissolved, and make up with water and phosphate to prepare a solution with a concentration of 0.1 g / mL and a pH of 7.4 to obtain a lyophilization protectant;

[0088] S2. Take the monovalent bulk of the inactivated H1N1 influenza virus vaccine (from the production workshop of this company), and dilute it with 0.02 mol / L PBS buffer to a dilution with a hemagglutinin content of 42 μg / mL and a hemagglutination titer of 1:768 to obtain Diluent A;

[0089] S3. Take 200 mL of the Diluent A obtained in Step S2, and add the lyophilization protectant obtained in Step S1 to obtain a reference standard solution with a hemagglutinin content of 28 μg / mL and a hemagglutination titer of 1:512;

[0090] S4. Aliquot the reference standard solution obtained in Step S3 into lyophilization vials, and place them in a -40 °C low-temperature refrigerator for pre-freezing for 4 h;

[0091] S5. When the freezing is complete, place the frozen product in a sealed vacuum container with a vacuum degree of 6 Pa, and place it in a freeze dryer at -52 °C for drying for 28 h to obtain a dry white powder. Take it out and immediately seal it with a cap, and store it at 8 °C or below for standby to obtain the reference standard for the hemagglutination titer of the H1N1 influenza virus;

[0092] S6. Calibrate the hemagglutination titer reference product of H1N1 influenza virus;

[0093] The calibration steps are as follows:

[0094] S61. Take the hemagglutination titer reference product of H1N1 influenza virus antigen prepared in step S5, and reconstitute it with 0.5 mL of sterile injection water to obtain the reconstituted hemagglutination titer reference product of H1N1 influenza virus antigen;

[0095] S62. Add 50 μL of sterile 0.9 wt% sodium chloride solution to each well of the hemagglutination plate;

[0096] S63. Add 50 μL of the reconstituted hemagglutination titer reference product of H1N1 influenza virus antigen to the first well of the hemagglutination plate, take out 50 μL from the first well and add it to the second well, pipette and mix well, and perform 2-fold serial dilutions in sequence until a negative result of red blood cell agglutination (100% non-agglutination of red blood cells) appears. Discard 50 μL after mixing the last well;

[0097] S64. Add 50 μL of 1v% chicken red blood cell suspension to each well from low concentration to high concentration. Tap the hemagglutination plate to fully mix the red blood cells with the hemagglutination titer reference product of H1N1 influenza virus antigen, and then let it stand;

[0098] S65. Take one well, first add 50 μL of sterile 0.9% sodium chloride solution, and then add 50 μL of 1v% chicken red blood cell suspension. This group of controls can exclude the non-specific agglutination of red blood cells themselves;

[0099] S66. Incubation: Incubate at 25 °C for 30 min;

[0100] S67. Observe the red blood cell agglutination results according to the content of Table 1. Take the previous positive result before the negative result (100% non-agglutination of red blood cells) as the determination endpoint, and calculate the hemagglutination titer value of the hemagglutination titer reference product of H1N1 influenza virus according to the content of Table 2; Calculate the average value based on 20 independent detection results obtained in 20 batches as the tentative target value, and use this tentative target value as the target value for quality control in the next month. After one month, collect the detection results of this month together with the previous 20 detection results, and calculate the cumulative mean value (the first month). Use this cumulative mean value as the target value for the next month; Repeat the above operation process for 5 consecutive months. Take the cumulative mean value calculated from the initial 20 detection results and all the data collected in 5 months as the hemagglutination titer standard value of the hemagglutination titer reference product of H1N1 influenza virus, that is, the T0 of the hemagglutination titer reference product of H1N1 influenza virus is 1:515.

[0101] Preparation Example 2 provides a reference product for the hemagglutination titer of H3N2 influenza virus. The difference in its preparation steps from Preparation Example 1 is that in step S2, the monovalent stock solution of H1N1 influenza virus split vaccine is replaced by the monovalent stock solution of H3N2 influenza virus split vaccine.

[0102] Preparation Example 2 provides a reference product for the hemagglutination titer of H3N2 influenza virus. The difference in its preparation steps from Preparation Example 1 is that the monovalent stock solution of H1N1 influenza virus split vaccine is replaced by the monovalent stock solution of H3N2 influenza virus split vaccine; the T0 of the reference product for the hemagglutination titer of H3N2 influenza virus is 1:503.

[0103] Preparation Example 3 provides a reference product for the hemagglutination titer of BV influenza virus. The difference in its preparation steps from Preparation Example 1 is as follows:

[0104] S2. Take the monovalent stock solution of BV influenza virus split vaccine (from the production workshop of our company), and dilute it with 0.02 mol / L PBS buffer to a dilution solution with a hemagglutinin content of 39 μg / mL and a hemagglutination titer of 1:2048 to obtain Dilution A;

[0105] S3. Take 200 mL of Dilution A obtained in step S2, and add the lyoprotectant obtained in step S1 to obtain a reference product solution with a hemagglutinin content of 26 μg / mL and a hemagglutination titer of 1:1536;

[0106] The T0 of the reference product for the hemagglutination titer of BV influenza virus is 1:1492.

[0107] Preparation Example 4 provides a reference product for the hemagglutination titer of BY influenza virus. The difference in its preparation steps from Preparation Example 1 is as follows:

[0108] S2. Take the monovalent stock solution of BY influenza virus split vaccine (from the production workshop of our company), and dilute it with 0.02 mol / L PBS buffer to a dilution solution with a hemagglutinin content of 36 μg / mL and a hemagglutination titer of 1:1024 to obtain Dilution A;

[0109] S3. Take 200 mL of Dilution A obtained in step S2, and add the lyoprotectant obtained in step S1 to obtain a reference product solution with a hemagglutinin content of 24 μg / mL and a hemagglutination titer of 1:768;

[0110] The T0 of the reference product for the hemagglutination titer of BY influenza virus is 1:786.

[0111] Examples

[0112] Examples 1-5 provide a method for detecting the hemagglutination titer of an H1N1 influenza virus antigen sample.

[0113] Example 1 provides a method for detecting the hemagglutination titer of an H1N1 influenza virus harvest fluid, and the steps are as follows:

[0114] S1. Lysis treatment

[0115] The H1N1 influenza virus antigen hemagglutination titer reference product is reconstituted with 0.5 mL of sterile injection water to obtain an H1N1 influenza virus antigen hemagglutination titer reference product solution;

[0116] 10 wt% Detergent (the volume ratio of the H1N1 influenza virus harvest fluid to 10 wt% Detergent is 9:1, and the volume ratio of the H1N1 influenza virus antigen hemagglutination titer reference product solution to 10 wt% Detergent is 9:1) is added to the H1N1 influenza virus harvest fluid and the H1N1 influenza virus antigen hemagglutination titer reference product solution respectively, and lysed for 30 min to obtain the lysed H1N1 influenza virus harvest fluid and the lysed H1N1 influenza virus antigen hemagglutination titer reference product solution.

[0117] S2. Hemagglutination titer detection

[0118] S21. Hemagglutination plate labeling: Add 50 μL of sterile 0.9 wt% sodium chloride solution to each well of the hemagglutination plate;

[0119] S22. Serial dilution: Add 50 μL of the lysed H1N1 influenza virus harvest fluid and the lysed H1N1 influenza virus antigen hemagglutination titer reference product solution to the first well of the hemagglutination plate respectively. Take 50 μL from the first well and add it to the second well, pipette and mix well, and perform a 2-fold serial dilution in sequence until a negative result of red blood cell agglutination (100% non-agglutination of red blood cells) appears. Discard 50 μL after mixing the last well;

[0120] S23. Add chicken red blood cell suspension: Add 50 μL of 1 v% chicken red blood cell suspension to each well from low concentration to high concentration. Tap the hemagglutination plate to make the red blood cells fully mix with the lysed H1N1 influenza virus harvest fluid and the lysed H1N1 influenza virus antigen hemagglutination titer reference product solution respectively, and then let it stand;

[0121] S24. Negative control to exclude non-specific agglutination of red blood cells themselves: Take one well, first add 50 μL of sterile 0.9 wt% sodium chloride solution, and then add 50 μL of 1 v% chicken red blood cell suspension. This group of controls can exclude non-specific agglutination of red blood cells themselves;

[0122] S25. Incubation: Incubate at 25 °C for 30 min;

[0123] S26. Calculate the hemagglutination titer detection value based on the hemagglutination result: According to the content of Table 1 and Table 2, the previous positive result before the negative result (100% non-agglutination of red blood cells) is taken as the determination endpoint, and the dilution multiple of the previous positive result before the negative result is determined. Calculate the hemagglutination titer detection value T of the H1N1 influenza virus harvest fluid according to the hemagglutination titer calculation method in Table 2 t and the hemagglutination titer detection value T of the H1N1 influenza virus antigen hemagglutination titer reference product s .

[0124] S3. Calculate the hemagglutination titer standard value of the H1N1 influenza virus harvest fluid

[0125] Take the ratio between the hemagglutination titer detection value T of the H1N1 influenza virus harvest fluid t and the hemagglutination titer detection value T of the H1N1 influenza virus antigen hemagglutination titer reference product s as the correction coefficient, and correct the detection value of the H1N1 influenza virus harvest fluid to obtain the hemagglutination titer standard value of the H1N1 influenza virus harvest fluid. The calculation formula is:

[0126]

[0127] Among them, T is the hemagglutination titer standard value of the H1N1 influenza virus harvest fluid;

[0128] T t is the hemagglutination titer detection value of the H1N1 influenza virus harvest fluid;

[0129] T s is the hemagglutination titer detection value of the H1N1 influenza virus antigen hemagglutination titer reference product;

[0130] T0 is the hemagglutination titer standard value of the H1N1 influenza virus antigen hemagglutination titer reference product (T0 = 1:515).

[0131] Note: The samples taken each time are measured in parallel 6 times, and the detection results of the hemagglutination titer of the H1N1 influenza virus harvest fluid are shown in Table 3

[0132] Table 3 Detection results of the hemagglutination titer of the H1N1 influenza virus harvest fluid

[0133]

[0134] Example 2 provides a method for detecting the hemagglutination titer of a deglycosylated and purified solution of H1N1 influenza virus. The difference from Example 1 is that the H1N1 influenza virus harvest solution is replaced with the deglycosylated and purified solution of H1N1 influenza virus in equal volume. The deglycosylated and purified solution of H1N1 influenza virus is diluted 40-fold with 0.02 mol / L PBS buffer before lysis to obtain a diluted solution of the deglycosylated and purified solution of H1N1 influenza virus. The volume ratio of the diluted solution of the deglycosylated and purified solution of H1N1 influenza virus to 10 wt% Detergent is 9:1. The standard value of the hemagglutination titer of the deglycosylated and purified solution of H1N1 influenza virus is calculated by the formula:

[0135]

[0136] where T is the standard value of the hemagglutination titer of the deglycosylated and purified solution of H1N1 influenza virus;

[0137] T t is the detected value of the hemagglutination titer of the diluted solution of the deglycosylated and purified solution of H1N1 influenza virus;

[0138] T s is the detected value of the hemagglutination titer of the H1N1 influenza virus antigen hemagglutination titer reference product;

[0139] T0 is the standard value of the hemagglutination titer of the H1N1 influenza virus antigen hemagglutination titer reference product (T0 = 1:515);

[0140] X is the dilution factor of the diluted solution of the deglycosylated and purified solution of H1N1 influenza virus (the value is 40).

[0141] The detection results of the hemagglutination titer of the deglycosylated and purified solution of H1N1 influenza virus are shown in Table 4.

[0142] Table 4 Detection Results of Hemagglutination Titer of Deglycosylated and Purified Solution of H1N1 Influenza Virus

[0143]

[0144] Example 3 provides a method for detecting the hemagglutination titer of an H1N1 influenza virus lysate. The difference from Example 2 is that the deglycosylated and purified solution of H1N1 influenza virus is replaced with the H1N1 influenza virus lysate in equal volume. The detection results of the hemagglutination titer of the H1N1 influenza virus lysate are shown in Table 5.

[0145] Table 5 Detection Results of Hemagglutination Titer of H1N1 Influenza Virus Lysate

[0146]

[0147] Example 4 provided a method for detecting the hemagglutination titer of the purified solution after lysis of H1N1 influenza virus. The difference from Example 2 was that the deglycosylated purified solution of H1N1 influenza virus was replaced with the purified solution after lysis of H1N1 influenza virus in equal volume. The purified solution after lysis of H1N1 influenza virus was diluted 15 times with 0.02 mol / L PBS buffer. The detection results of the hemagglutination titer of the purified solution after lysis of H1N1 influenza virus were shown in Table 6.

[0148] Table 6 Detection Results of Hemagglutination Titer of Purified Solution after Lysis of H1N1 Influenza Virus

[0149]

[0150] Example 5 provided a method for detecting the hemagglutination titer of the monovalent stock solution of H1N1 influenza virus. The difference from Example 2 was that the deglycosylated purified solution of H1N1 influenza virus was replaced with the monovalent stock solution of H1N1 influenza virus in equal volume. The dilution factor of the monovalent stock solution of H1N1 influenza virus was 15. The detection results of the hemagglutination titer of the monovalent stock solution of H1N1 influenza virus were shown in Table 7.

[0151] Table 7 Detection Results of Hemagglutination Titer of Monovalent Stock Solution of H1N1 Influenza Virus

[0152]

[0153]

[0154] As can be seen from Tables 3 - 7, the method for detecting the hemagglutination titer of the H1N1 influenza virus antigen sample provided in this application had good precision and repeatability, and the RSD was less than 15%. This method was stable and accurate for detecting the harvest fluid, deglycosylated purified solution, lysis solution, purified solution after lysis, and monovalent stock solution of H1N1 influenza virus, and could be used for the quality control evaluation of intermediates in each process during the production of the monovalent stock solution of H1N1 influenza virus split vaccine.

[0155] Examples 6 - 10 provided a method for detecting the hemagglutination titer of the H3N2 influenza virus antigen sample.

[0156] Example 6 provided a method for detecting the hemagglutination titer of the harvest fluid of H3N2 influenza virus. The difference from Example 1 was as follows:

[0157] The harvest fluid of H1N1 influenza virus was replaced with the harvest fluid of H3N2 influenza virus in equal volume;

[0158] The reference product for the hemagglutination titer of H1N1 influenza virus antigen was replaced with the reference product for the hemagglutination titer of H3N2 influenza virus antigen in equal mass. The standard value of the hemagglutination titer of the reference product for the hemagglutination titer of H3N2 influenza virus antigen was (T0 = 1:503).

[0159] The test results of the hemagglutination titer of the harvested solution of H3N2 influenza virus are shown in Table 8.

[0160] Table 8 Test results of the hemagglutination titer of the harvested solution of H3N2 influenza virus

[0161]

[0162] Example 7 provides a method for detecting the hemagglutination titer of the deglycosylated and purified solution of H3N2 influenza virus. The difference from Example 2 is that:

[0163] The deglycosylated and purified solution of H1N1 influenza virus is replaced with the deglycosylated and purified solution of H3N2 influenza virus in equal volume;

[0164] The reference product of the hemagglutination titer of H1N1 influenza virus antigen is replaced with the reference product of the hemagglutination titer of H3N2 influenza virus antigen in equal mass. The standard value of the hemagglutination titer of the reference product of H3N2 influenza virus antigen is (T0 = 1:503);

[0165] The dilution factor of the deglycosylated and purified solution of H3N2 influenza virus is 25.

[0166] The test results of the hemagglutination titer of the deglycosylated and purified solution of H3N2 influenza virus are shown in Table 9.

[0167] Table 9 Test results of the hemagglutination titer of the deglycosylated and purified solution of H3N2 influenza virus

[0168]

[0169]

[0170] Example 8 provides a method for detecting the hemagglutination titer of the lysate of H3N2 influenza virus. The difference from Example 7 is that the deglycosylated and purified solution of H3N2 influenza virus is replaced with the lysate of H3N2 influenza virus in equal volume.

[0171] The test results of the hemagglutination titer of the lysate of H3N2 influenza virus are shown in Table 10.

[0172] Table 10 Test results of the hemagglutination titer of the lysate of H3N2 influenza virus

[0173]

[0174] Example 9 provides a method for detecting the hemagglutination titer of the purified solution after lysis of H3N2 influenza virus. The difference from Example 7 is that:

[0175] The deglycosylated and purified solution of H3N2 influenza virus is replaced with the purified solution after lysis of H3N2 influenza virus in equal volume; the dilution factor of the purified solution after lysis of H3N2 influenza virus is 10.

[0176] The detection results of the hemagglutination titer of the purified solution after lysis of H3N2 influenza virus are shown in Table 11.

[0177] Table 11 Detection results of the hemagglutination titer of the purified solution after lysis of H3N2 influenza virus

[0178]

[0179] Example 10 provides a method for detecting the hemagglutination titer of the monovalent stock solution of H3N2 influenza virus. The difference from Example 7 is as follows:

[0180] The deglycosylated purified solution of H3N2 influenza virus is replaced with the monovalent stock solution of H3N2 influenza virus in equal volume; the dilution factor of the monovalent stock solution of H3N2 influenza virus is 10.

[0181] The detection results of the hemagglutination titer of the monovalent stock solution of H3N2 influenza virus are shown in Table 12.

[0182] Table 12 Detection results of the hemagglutination titer of the monovalent stock solution of H3N2 influenza virus

[0183]

[0184]

[0185] As can be seen from Tables 8 - 12, the method for detecting the hemagglutination titer of the H3N2 influenza virus antigen sample provided in this application has good precision and repeatability, with the RSD all less than 15%. This method is stable and accurate for detecting the harvest fluid, deglycosylated purified solution, lysate, purified solution after lysis, and monovalent stock solution of H3N2 influenza virus, and can be used for the quality control evaluation of intermediates in each process during the production of the monovalent stock solution of H3N2 influenza virus split vaccine.

[0186] Examples 11 - 15 provide a method for detecting the hemagglutination titer of a BV influenza virus antigen sample.

[0187] Example 11 provides a method for detecting the hemagglutination titer of the harvest fluid of BV influenza virus. The difference from Example 1 is as follows:

[0188] The harvest fluid of H1N1 influenza virus is replaced with the harvest fluid of BV influenza virus in equal volume;

[0189] The reference product for the hemagglutination titer of H1N1 influenza virus antigen is replaced with the reference product for the hemagglutination titer of BV influenza virus antigen in equal mass. The standard value of the hemagglutination titer of the reference product for the hemagglutination titer of BV influenza virus is (T0 = 1:1492).

[0190] The detection results of the hemagglutination titer of the harvest fluid of BV influenza virus are shown in Table 13.

[0191] Table 13 Detection Results of Hemagglutination Titers of Harvest Fluids of Influenza B / Victoria Virus

[0192]

[0193] Example 12 provides a method for detecting the hemagglutination titer of a deglycosylated and purified fluid of influenza B / Victoria virus. The difference from Example 2 is that:

[0194] The deglycosylated and purified fluid of influenza A / H1N1 virus is replaced with an equal volume of the deglycosylated and purified fluid of influenza B / Victoria virus;

[0195] The reference product of hemagglutination titer of influenza A / H1N1 virus antigen is replaced with an equal mass of the reference product of hemagglutination titer of influenza B / Victoria virus antigen, and the standard value of the hemagglutination titer of the reference product of hemagglutination titer of influenza B / Victoria virus antigen is (T0 = 1:1492);

[0196] The dilution factor of the deglycosylated and purified fluid of influenza B / Victoria virus is 25.

[0197] The detection results of the hemagglutination titer of the deglycosylated and purified fluid of influenza B / Victoria virus are shown in Table 14.

[0198] Table 14 Detection Results of Hemagglutination Titers of Deglycosylated and Purified Fluids of Influenza B / Victoria Virus

[0199]

[0200] Example 13 provides a method for detecting the hemagglutination titer of a lysate of influenza B / Victoria virus. The difference from Example 12 is that: the deglycosylated and purified fluid of influenza B / Victoria virus is replaced with an equal volume of the lysate of influenza B / Victoria virus.

[0201] The detection results of the hemagglutination titer of the lysate of influenza B / Victoria virus are shown in Table 15.

[0202] Table 15 Detection Results of Hemagglutination Titers of Lysates of Influenza B / Victoria Virus

[0203]

[0204] Example 14 provides a method for detecting the hemagglutination titer of a purified fluid after lysis of influenza B / Victoria virus. The difference from Example 12 is that:

[0205] The deglycosylated and purified fluid of influenza B / Victoria virus is replaced with an equal volume of the purified fluid after lysis of influenza B / Victoria virus; the dilution factor of the purified fluid after lysis of influenza B / Victoria virus is 8.

[0206] The detection results of the hemagglutination titer of the purified fluid after lysis of influenza B / Victoria virus are shown in Table 16.

[0207] Table 16 Detection Results of Hemagglutination Titers of Purified Fluids after Lysis of Influenza B / Victoria Virus

[0208]

[0209] Example 15 provides a method for detecting the hemagglutination titer of a monovalent bulk of influenza virus type BV, which is different from Example 12 in that:

[0210] The deglycosylated and purified solution of influenza virus type BV is replaced with an equal volume of the monovalent bulk of influenza virus type BV; the dilution factor of the monovalent bulk of influenza virus type BV is 6.

[0211] The detection results of the hemagglutination titer of the monovalent bulk of influenza virus type BV are shown in Table 17.

[0212] Table 17 Detection Results of the Hemagglutination Titer of the Monovalent Bulk of Influenza Virus Type BV

[0213]

[0214] As can be seen from Tables 13 - 17, the method for detecting the hemagglutination titer of the influenza virus type BV antigen sample provided in this application has good precision and repeatability, and the RSDs are all less than 15%. This method can stably and accurately detect the harvest fluid, deglycosylated and purified solution, lysate, purified solution after lysis, and monovalent bulk of influenza virus type BV, and can be used for the quality control evaluation of intermediates in each process during the production of the monovalent bulk of the split vaccine of influenza virus type BV.

[0215] Examples 16 - 20 provide a method for detecting the hemagglutination titer of an influenza virus type BY antigen sample.

[0216] Example 16 provides a method for detecting the hemagglutination titer of the harvest fluid of influenza virus type BY, which is different from Example 1 in that:

[0217] The harvest fluid of influenza virus type H1N1 is replaced with an equal volume of the harvest fluid of influenza virus type BY;

[0218] The hemagglutination titer reference product of influenza virus type H1N1 antigen is replaced with an equal mass of the hemagglutination titer reference product of influenza virus type BY, and the standard value of the hemagglutination titer of the hemagglutination titer reference product of influenza virus type BY is (T0 = 1:786).

[0219] The detection results of the hemagglutination titer of the harvest fluid of influenza virus type BY are shown in Table 18.

[0220] Table 18 Detection Results of the Hemagglutination Titer of the Harvest Fluid of Influenza Virus Type BY

[0221]

[0222] Example 17 provides a method for detecting the hemagglutination titer of the deglycosylated and purified solution of influenza virus type BY, which is different from Example 2 in that:

[0223] Replace the deglycosylated and purified solution of H1N1 influenza virus with an equal volume of the deglycosylated and purified solution of BY influenza virus;

[0224] Replace the H1N1 influenza virus antigen hemagglutination titer reference product with an equal mass of the BY influenza virus antigen hemagglutination titer reference product. The standard value of the hemagglutination titer of the BY influenza virus antigen hemagglutination titer reference product is (T0 = 1:786);

[0225] The dilution factor of the deglycosylated and purified solution of BY influenza virus is 40.

[0226] The detection results of the hemagglutination titer of the deglycosylated and purified solution of BY influenza virus are shown in Table 19.

[0227] Table 19 Detection results of the hemagglutination titer of the deglycosylated and purified solution of BY influenza virus

[0228]

[0229] Example 18 provides a method for detecting the hemagglutination titer of a BY influenza virus lysate, which is different from Example 17 in that: the deglycosylated and purified solution of BY influenza virus is replaced with an equal volume of the BY influenza virus lysate.

[0230] The detection results of the hemagglutination titer of the BY influenza virus lysate are shown in Table 20.

[0231] Table 20 Detection results of the hemagglutination titer of the BY influenza virus lysate

[0232]

[0233] Example 19 provides a method for detecting the hemagglutination titer of a purified solution after lysis of BY influenza virus, which is different from Example 17 in that:

[0234] The deglycosylated and purified solution of BY influenza virus is replaced with an equal volume of the purified solution after lysis of BY influenza virus; the dilution factor of the purified solution after lysis of BY influenza virus is 10.

[0235] The detection results of the hemagglutination titer of the purified solution after lysis of BY influenza virus are shown in Table 21.

[0236] Table 21 Detection results of the hemagglutination titer of the purified solution after lysis of BY influenza virus

[0237]

[0238] Example 20 provides a method for detecting the hemagglutination titer of a monovalent stock solution of BY influenza virus, which is different from Example 17 in that:

[0239] The de - sugared and purified solution of influenza virus type BY is replaced with the monovalent stock solution of influenza virus type BY in equal volume; the dilution factor of the monovalent stock solution of influenza virus type BY is 10.

[0240] The detection results of the hemagglutination titer of the monovalent stock solution of influenza virus type BY are shown in Table 22.

[0241] Table 22 Detection results of the hemagglutination titer of the monovalent stock solution of influenza virus type BY

[0242]

[0243] As can be seen from Tables 18 - 22, the detection method for the hemagglutination titer of the influenza virus type BY antigen sample provided in this application has good precision and repeatability, with RSD values all less than 15%. This method can stably and accurately detect the harvest fluid, de - sugared and purified fluid, lysate, post - lysate purified fluid, and monovalent stock solution of influenza virus type BY, and can be used for the quality control and evaluation of intermediates in each process during the production of the monovalent stock solution of the split vaccine of influenza virus type BY.

[0244] Example 21, which is different from Example 5 in that the influence of the storage days of the monovalent stock solution of influenza virus type H1N1 on the hemagglutination titer is investigated, and the test results are shown in Table 23.

[0245] Table 23 Detection results of the hemagglutination titer of the monovalent stock solution of influenza virus type H1N1 on different days

[0246]

[0247] As can be seen from Table 23, the detection method for the hemagglutination titer of the influenza virus antigen sample provided in this application has high detection accuracy, and factors such as storage time have little influence on the detection results, and the stability of the detection results is good.

[0248] Accuracy analysis of the hemagglutination titer detection method

[0249] The hemagglutination titers of influenza virus antigen hemagglutination titer reference products with different concentrations are measured, and the recovery rate is calculated (recovery rate = measured value / true value × 100%), and the accuracy of the hemagglutination titer detection method is analyzed based on the recovery rate.

[0250] Example 22, which is different from Example 5 in that the monovalent stock solution of influenza virus type H1N1 is replaced with the H1N1 influenza virus hemagglutination titer reference product prepared in Preparation Example 1 in equal volume. The hemagglutination titer and recovery rate of the H1N1 influenza virus hemagglutination titer reference product are shown in Table 24.

[0251] Table 24 Hemagglutination titer and recovery rate of the H1N1 influenza virus hemagglutination titer reference product

[0252]

[0253] Example 23, which is different from Example 22 in that the concentration of the reference product for the hemagglutination titer of H1N1 influenza virus prepared in Preparation Example 1 is replaced by 80%, and the hemagglutination titer and recovery rate of the reference product for the hemagglutination titer of H1N1 influenza virus at a concentration of 80% are shown in Table 25.

[0254] Table 25 Hemagglutination titer and recovery rate of the reference product for the hemagglutination titer of H1N1 influenza virus at a concentration of 80%

[0255]

[0256] As can be seen from Tables 24-25, the method for detecting the hemagglutination titer of the influenza virus antigen sample provided by the present application can accurately analyze the hemagglutination titer of the influenza virus antigen sample, and the accuracy is relatively high.

[0257] This specific embodiment is only an explanation of the present application, and it is not a limitation to the present application. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A method for detecting the hemagglutination titer of an influenza virus antigen sample, characterized in that, It includes the following steps: S1. Lysis treatment: Add a lysis agent to the influenza virus antigen sample and the influenza virus antigen hemagglutination titer reference product respectively for lysis to obtain a lysed influenza virus antigen sample and a lysed influenza virus antigen hemagglutination titer reference product; the influenza virus antigen sample is an intermediate produced in each process during the production of the monovalent bulk of the influenza virus split vaccine; the hemagglutinin content of the influenza virus antigen sample before lysis is 10 - 50 μg / mL; the influenza virus antigen hemagglutination titer reference product needs to be reconstituted with 0.5 mL of sterile injection water before adding the lysis agent to obtain a solution of the influenza virus antigen hemagglutination titer reference product; S2. Hemagglutination titer detection: Detect the hemagglutination titers of the lysed influenza virus antigen sample and the lysed influenza virus antigen hemagglutination titer reference product respectively to obtain the hemagglutination titer detection value of the influenza virus antigen sample and the hemagglutination titer detection value of the influenza virus antigen hemagglutination titer reference product; S3. Calculate the hemagglutination titer standard value of the influenza virus antigen sample: Use the ratio between the hemagglutination titer standard value of the influenza virus antigen hemagglutination titer reference product and the hemagglutination titer detection value of the influenza virus antigen hemagglutination titer reference product as a correction factor to correct the hemagglutination titer detection value of the influenza virus antigen sample to obtain the hemagglutination titer standard value of the influenza virus antigen sample.

2. The detection method of the hemagglutination titer of an influenza virus antigen sample according to claim 1, wherein, The influenza virus antigen sample includes any one of the influenza virus harvest fluid, the influenza virus deglycosylated and purified fluid, the influenza virus lysate, the influenza virus post-lysis purified fluid, or the monovalent bulk of the influenza virus split vaccine.

3. The detection method of the hemagglutination titer of an influenza virus antigen sample according to claim 2, wherein The monovalent bulk of the influenza virus split vaccine includes any one of the monovalent bulk of H1N1 influenza virus, the monovalent bulk of H3N2 influenza virus, the monovalent bulk of BV influenza virus, or the monovalent bulk of BY influenza virus.

4. The detection method of the hemagglutination titer of an influenza virus antigen sample according to claim 3, characterized in that, The lysis agent is selected from any one of Triton X-100, polysorbate 80, Triton N101 or any one of the detergents in 3-14Detergent.

5. The detection method of the hemagglutination titer of an influenza virus antigen sample according to claim 4, characterized in that, In the step S1, the lysing agent is 10 wt% 3-14 Detergent.

6. The detection method of the hemagglutination titer of an influenza virus antigen sample according to claim 5, characterized in that, In the S1 step, the volume ratio of the influenza virus antigen sample to the lysis agent before lysis and the volume ratio of the solution of the influenza virus antigen hemagglutination titer reference product to the lysis agent are both (8 - 10):

1.

7. The detection method of the hemagglutination titer of an influenza virus antigen sample according to claim 6, wherein In the S1 step, the time of the lysis treatment is 20 - 40 min.

8. The detection method of the hemagglutination titer of an influenza virus antigen sample according to claim 1, characterized in that, In the S2 step, the specific steps of the hemagglutination titer detection include: S21. Mark the hemagglutination plate; S22. Serial dilution: Add 50 μL of the lysed influenza virus antigen sample and the lysed influenza virus antigen hemagglutination titer reference product to the first well of the hemagglutination plate respectively. Take out 50 μL from the first well and add it to the second well, pipette and mix well, and perform a 2-fold serial dilution in sequence until a negative result of red blood cell agglutination appears. Discard 50 μL after mixing the last well; S23. Add chicken red blood cell suspension; S24. Exclude the non-specific agglutination of red blood cells by the negative control; S25. Incubate; S26. Calculate the hemagglutination titer detection value: Observe the red blood cell agglutination result, take the previous positive before the negative result appears as the determination end point, and calculate the hemagglutination titer detection value of the influenza virus antigen sample and the hemagglutination titer detection value of the influenza virus antigen hemagglutination titer reference product respectively.

9. A method for detecting the hemagglutination titer of an influenza virus antigen sample according to any one of claims 1 to 8, characterized in that, In the S3 step, the calculation formula for the hemagglutination titer standard value of the influenza virus antigen sample is: Where, T is the hemagglutination titer standard value of the influenza virus antigen sample; T t is the hemagglutination titer detection value of the influenza virus antigen sample; T s It is the hemagglutination titer detection value of the influenza virus antigen hemagglutination titer reference product; T0 is the standard value of the hemagglutination titer of the influenza virus antigen hemagglutination titer reference preparation.

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