CN114184797B - A method for detecting hemagglutinin in monovalent stock solution of influenza virus split vaccine - Google Patents

Abstract

The application discloses a method for detecting influenza virus split vaccine monovalent stock solution hemagglutinin, which comprises the following steps: s1, respectively adding a cracking agent into an influenza virus cracked vaccine monovalent stock solution and an influenza virus antigen reference for cracking; s2, respectively detecting the hemagglutination titer of the split influenza virus split vaccine monovalent stock solution and the hemagglutination titer of the split influenza virus antigen reference; s3, calculating the standard value of the hemagglutination titer of the monovalent stock solution of the influenza virus split vaccine according to the detection value of the hemagglutination titer of the monovalent stock solution of the influenza virus split vaccine; s4, calculating the hemagglutinin standard value of the monovalent stock solution of the influenza virus split vaccine according to the hemagglutinin standard value of the monovalent stock solution of the influenza virus split vaccine. The detection method provided by the application improves the stability, accuracy and sensitivity of the detection result, shortens the detection time from 2 days to less than 2 hours, and accelerates the production progress of influenza vaccine.

Description

A method for detecting hemagglutinin in monovalent stock solution of influenza virus split vaccine

Technical Field

The invention belongs to the field of biotechnology and relates to a method for detecting biological preparations, in particular to a method for detecting hemagglutinin in a monovalent stock solution of an influenza virus split vaccine.

Background technique

Hemagglutinin is a glycoprotein on the surface of influenza virus. Hemagglutinin can induce protective immune response in a variety of hosts and is currently the only marker for the efficacy of influenza vaccine. Therefore, how to quickly and accurately measure the hemagglutinin of influenza vaccine can evaluate the efficacy of influenza vaccine and also play a positive role in preventing influenza virus infection.

The principle of conventional one-way immunodiffusion method for detecting influenza virus hemagglutinin is: antigen-antibody specific binding forms a precipitation ring, and the diameter of the precipitation ring is measured after staining. The method for detecting influenza virus hemagglutinin by conventional one-way immunodiffusion method includes the following steps: 1) agarose preparation and punching; 2) antigen standard solution preparation; 3) antigen standard and influenza virus sample dilution and lysis; 4) sample loading and diffusion; 5) gel washing; 6) glue pressing; 7) drying; 8) staining and decolorization; 9) determination of precipitation ring diameter; 10) calculation of the hemagglutinin content of influenza virus sample. However, the conventional one-way immunodiffusion method for detecting influenza virus hemagglutinin has the following shortcomings: 1) factors such as the concentration of agarose used for detection, the toughness of the gel plate, the staining time, the decolorization time, the shape and depth of the precipitation ring affect the results of the hemagglutination test, resulting in low test accuracy; 2) the test time often takes 2 days, the test cycle is long, and the test efficiency is low; 3) the amount of antigen standard is large, and there may be a shortage of antigen standard, which is less economical.

In order to effectively improve the above-mentioned deficiencies of conventional one-way immunodiffusion method for detecting influenza virus hemagglutinin, the present application provides a detection method for detecting influenza virus hemagglutinin that replaces the conventional one-way immunodiffusion method.

Summary of the invention

In order to effectively improve the shortcomings of conventional one-way immunodiffusion method for detecting influenza virus hemagglutinin and improve the accuracy and efficiency of influenza virus hemagglutinin detection method, the present application provides a method for detecting hemagglutinin of monovalent stock solution of influenza virus split vaccine.

In the first aspect, the present application provides a method for detecting hemagglutinin in a monovalent stock solution of an influenza virus split vaccine, which is implemented using the following technical solution:

A method for detecting hemagglutinin in monovalent stock solution of influenza virus split vaccine comprises the following steps:

S1. Splitting treatment: adding a splitting agent to the influenza virus split vaccine monovalent stock solution and the influenza virus antigen reference substance respectively for splitting, thereby obtaining a split influenza virus split vaccine monovalent stock solution and a split influenza virus antigen reference substance;

S2. Hemagglutination titer detection: respectively detecting the hemagglutination titer of the split influenza virus split vaccine monovalent stock solution and the hemagglutination titer of the split influenza virus antigen reference substance, and obtaining the hemagglutination titer detection value of the influenza virus split vaccine monovalent stock solution and the hemagglutination titer detection value of the influenza virus antigen reference substance;

S3. Calculation of the standard value of the hemagglutination titer of the monovalent stock solution of the influenza virus split vaccine: The standard value of the hemagglutination titer of the monovalent stock solution of the influenza virus split vaccine is calculated from the hemagglutination titer detection value of the monovalent stock solution of the influenza virus split vaccine. The calculation formula is shown in Formula 1:

Wherein, T is the standard value of the hemagglutination titer of the monovalent stock solution of influenza virus split vaccine;

T _t is the hemagglutination titer test value of the monovalent stock solution of influenza virus split vaccine;

_Ts is the hemagglutination titer test value of the influenza virus antigen reference;

T ₀ is the standard value of the hemagglutination titer of the influenza virus antigen reference;

S4. Calculation of the hemagglutinin standard value of the monovalent stock solution of the influenza virus split vaccine: The hemagglutinin standard value of the monovalent stock solution of the influenza virus split vaccine is calculated from the hemagglutination titer standard value of the monovalent stock solution of the influenza virus split vaccine. The calculation formula is shown in Formula 2:

Wherein, C is the standard value of hemagglutinin of the monovalent stock solution of influenza virus split vaccine;

T is the standard value of the hemagglutination titer of the monovalent stock solution of influenza virus split vaccine;

T ₀ is the standard value of the hemagglutination titer of the influenza virus antigen reference;

_C0 is the standard value of hemagglutinin of influenza virus antigen reference.

By adopting the above technical scheme, the present application splits the monovalent stock solution of influenza virus split vaccine, and uses influenza virus antigen reference materials in the detection process, effectively eliminating the influence of factors such as chicken blood cell quality, concentration, storage time on the test results, and reducing the influence of the operation process on the test results, significantly improving the stability of the test results. At the same time, the detection method of the monovalent stock solution of hemagglutinin of influenza virus split vaccine in the present application can effectively avoid the shortcomings of the conventional one-way immune diffusion method, specifically: on the one hand, the present application avoids the influence of factors such as agarose concentration, gel plate toughness, staining time, decolorization time, shape and depth of the precipitation ring on the test results of hemagglutinin, and improves the stability, accuracy and sensitivity of the test results; on the other hand, the present application shortens the inspection time from 2 days to less than 2 hours, speeds up the inspection, and can publish the test results in time, thereby speeding up the production progress of influenza vaccine; and the present application effectively alleviates the problem of expensive international standard products for conventional one-way immune diffusion detection, further speeding up the production progress of influenza vaccine, and is suitable for the quality control evaluation of monovalent stock solution of influenza virus split vaccine.

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

In the present application, the production process of the influenza virus split vaccine monovalent stock solution includes the following steps:

(1) inoculating the diluted working seed (influenza virus) into the allantoic cavity of a chicken embryo and culturing for 48 to 72 hours; after culturing, screening the live chicken embryos, harvesting the allantoic sacs in a container, and testing the allantoic harvested fluid in each harvesting container, combining the allantoic fluids that have passed the test to obtain a monovalent influenza virus combined fluid; adding formaldehyde at a concentration of no more than 200 μg/mL to the monovalent influenza virus combined fluid to inactivate the virus, and obtaining an inactivated monovalent influenza virus combined fluid; centrifuging the inactivated monovalent influenza virus combined fluid and concentrating it by ultrafiltration to obtain an influenza virus harvested fluid;

(2) purifying the influenza virus harvested fluid by sucrose density gradient centrifugation to obtain influenza virus desugared purified fluid;

(3) adding a lysing agent to the influenza virus desugared purified solution to perform virus lysing to obtain an influenza virus lysate;

(4) repurifying the influenza virus lysate by column chromatography or sucrose density gradient centrifugation to obtain a purified influenza virus lysate;

(5) Sterilize the purified solution after influenza virus splitting to obtain a monovalent stock solution of influenza virus split vaccine.

The method for detecting hemagglutinin of monovalent stock solution of influenza virus split vaccine provided in the present application has excellent detection effect on hemagglutinin of various influenza viruses, and has high detection stability, accuracy and sensitivity. The detection results can be used to evaluate the quality control of monovalent stock solution of H1N1 influenza virus, monovalent stock solution of H3N2 influenza virus, monovalent stock solution of BV influenza virus or monovalent stock solution of BY influenza virus.

Preferably, in step S1, the influenza virus split vaccine monovalent stock solution needs to be diluted with PBS buffer to an influenza virus split vaccine antigen content of 10 to 50 μg/mL before adding the lysing agent to obtain an influenza virus split vaccine monovalent stock solution dilution; the influenza virus antigen reference substance needs to be reconstituted with sterile water for injection before adding the lysing agent to obtain an influenza virus antigen reference substance solution.

The present application dilutes the influenza virus split vaccine monovalent stock solution and the influenza virus antigen reference substance, which is beneficial to the subsequent lysis and can improve the lysis effect.

Preferably, in step S1, the volume ratio of the influenza virus split vaccine monovalent stock solution diluent to the lysing agent, and the volume ratio of the influenza virus antigen reference solution to the lysing agent are both (8-10):1.

The present application controls the volume ratio of the influenza virus split vaccine monovalent stock solution to the lysis agent, and the volume ratio of the influenza virus antigen reference solution to the lysis agent to be (8-10):1, so that the influenza virus split vaccine monovalent stock solution and the influenza virus antigen reference can be completely lysed, effectively eliminating the influence of factors such as chicken blood cell quality, concentration, storage time, etc. on the test results, and reducing the influence of the operation process on the test results, and significantly improving the stability of the test results.

Preferably, in the step S1, the cleavage treatment time is 20 to 40 minutes.

More preferably, in the step S1, the cleavage treatment time is 30 minutes.

The present application controls the lysis time to be 20 to 40 minutes, can effectively lyse the influenza virus lysate vaccine monovalent stock solution and the influenza virus antigen reference material, and has a short lysis time and a fast lysis rate, among which 30 minutes is better in terms of efficiency and lysis effect.

Preferably, the lysing agent is selected from Triton X-100, polysorbate 80, Triton N101 or Any one of 3-14 Detergents.

More preferably, the cleavage agent is 3-14Detergent.

This application was found in the research 3-14Detergent has excellent lysis effects on influenza virus split vaccine monovalent stock solution and influenza virus antigen reference material. It can effectively eliminate the influence of factors such as chicken blood cell quality, concentration, storage time, etc. on the test results, and reduce the influence of the operation process on the test results, significantly improving the stability of the test results.

Preferably, the The concentration of 3-14 Detergent is 10wt%.

Mass fraction of 10wt% 3-14 Detergent has an appropriate concentration and has a better lysis effect on the influenza virus split vaccine monovalent stock solution and influenza virus antigen reference product, and can completely lyse them.

Preferably, in step S1, the influenza virus split vaccine monovalent stock solution dilution is mixed with 3-14 Volume ratio of Detergent, influenza virus antigen reference solution and /> The volume ratio of 3-14 Detergent is 9:1.

Preferably, in step S2, the specific steps of the hemagglutination titer detection include:

S21, blood coagulation plate marker;

S22, serial dilution;

S23, adding chicken red blood cell suspension;

S24, negative control to exclude nonspecific agglutination of red blood cells themselves;

S25, incubation;

S26. Calculate the hemagglutination titer test value based on the red blood cell agglutination result.

Preferably, the operation steps of step S26 include:

Observe the results of red blood cell agglutination, take the previous positive result as the judgment end point, and calculate the hemagglutination titer detection value of the influenza virus split vaccine monovalent stock solution and the hemagglutination titer detection value of the influenza virus antigen reference respectively.

In the present application, the hematocrit results are specifically shown in Table 1.

Table 1 Red blood cell agglutination results

As can be seen from Table 1, in this application, the appearance of a negative result refers to 100% non-agglutination of red blood cells, that is, the phenomenon corresponding to "-". The previous positive of a negative result refers to the phenomenon before the appearance of "-", that is, it may be one of the phenomena corresponding to "++++", "+++", "++" or "+".

Table 2 Calculation method of hemagglutination titer

result Hemagglutination titer calculation method ++++ 1: (the dilution factor of the previous positive result of the negative result × 3/2) +++ 1: (the dilution factor of the previous positive result of the negative result × 5/4) ++ 1: The dilution multiple of the previous positive result of the negative result + 1: (the dilution factor of the previous positive result of the negative result × 7/8)

In summary, this application has the following beneficial effects:

1. The present application lyses the monovalent stock solution of influenza virus split vaccine and uses influenza virus antigen reference materials during the detection process, which effectively eliminates the influence of factors such as chicken blood cell quality, concentration, storage time, etc. on the test results, and reduces the influence of the operation process on the test results, thereby significantly improving the stability of the test results.

2. The present application discloses a method for detecting hemagglutinin in monovalent stock solution of influenza virus split vaccine. On the one hand, the present application avoids the influence of factors such as agarose concentration, toughness of gel plate, staining time, decolorization time, shape and depth of precipitation ring on the hemagglutinin detection result, thereby improving the stability, accuracy and sensitivity of the detection result. On the other hand, the present application shortens the inspection time from 2 days to less than 2 hours, speeds up the inspection, and can publish the test results in time, thereby accelerating the production progress of influenza vaccine.

3. The method for detecting hemagglutinin in monovalent stock solution of influenza virus split vaccine of the present application effectively alleviates the problem of expensive international standard products used in conventional one-way immunodiffusion method detection, further accelerates the production progress of influenza vaccine, and is suitable for quality control evaluation of monovalent stock solution of influenza virus split vaccine.

Detailed ways

The present application is further described in detail below with reference to the embodiments.

The lysing agent used in this application can be obtained commercially, wherein: 3-14 Detergent was purchased from EMD Milipore.

The monovalent stock solution of influenza virus split vaccine is a sample prepared in the production process, and three batches of samples were taken. Among them, the three batches of monovalent stock solution of H1N1 influenza virus split vaccine were recorded as A1-1, A1-2, and A1-3 respectively;

The three batches of sampling of the monovalent stock solution of H3N2 influenza virus split vaccine were recorded as A3-1, A3-2, and A3-3;

The three batches of samples of the monovalent stock solution of the BV influenza virus split vaccine were recorded as BV-1, BV-2, and BV-3;

The three batches of sampling of BY-type influenza virus split vaccine monovalent stock solution were respectively recorded as BY-1, BY-2, and BY-3.

The influenza virus antigen reference materials used in this application were all homemade.

Preparation Example 1 provides an H1N1 influenza virus antigen reference substance, the preparation steps of which are as follows:

S1. Add 30 mL of water to a container, heat to boil, add 10 g of maltose, stir until completely dissolved, add water and phosphate to prepare a 0.1 g/mL solution with a pH of 7.4 to obtain a lyophilization protective agent;

S2. Take the monovalent stock solution of H1N1 influenza virus split vaccine (obtained from the company's production workshop) and dilute it with 0.02 mol/L PBS buffer to a dilution solution A with a hemagglutinin content of 42 μg/mL;

S3, taking 200 mL of the dilution solution A obtained in step S2, adding the lyophilization protective agent obtained in step S1, and obtaining a reference solution with a hemagglutinin content of 28 μg/mL;

S4, the reference solution obtained in step S3 is divided into freeze-dried bottles and placed in a -40°C low-temperature refrigerator for pre-freezing for 4 hours;

S5. When the product is completely frozen, place it in a sealed vacuum container with a vacuum degree of 6 Pa, and place it in a freeze dryer at -52°C for 28 hours to obtain a dry white powder. After taking it out, immediately cover and seal it, and store it below 8°C for later use to obtain an H1N1 influenza virus antigen reference product;

S6. Calibrate the H1N1 influenza virus antigen reference material;

The calibration steps of the H1N1 influenza virus antigen reference hemagglutinin are as follows:

S61, taking the H1N1 influenza virus antigen reference substance prepared in step S5, and reconstituted it with 0.5 mL of sterile water for injection to obtain the reconstituted H1N1 influenza virus antigen reference substance;

S62. Add H1N1 influenza virus standard antiserum (batch number 19/314, purchased from NIBSC) to 25 mL of 1.5 wt% agarose solution at 56°C, and shake by hand (shake gently to avoid generating a large number of bubbles) to prepare solution A with a content of 35 μL/mL of H1N1 influenza virus standard antiserum; pour agarose on a gel plate, and quickly remove bubbles with a pipette if any; let stand until the agarose is completely solidified, and then put it in a 5°C refrigerator for 20 min. Take out the refrigerated agar plate and use a hole puncher to punch holes with a hole diameter of 3 mm and a distance between holes of 1.5 cm. Punch 36 holes in a 10 cm×10 cm agar plate, and use a vacuum pump to carefully suck out the gel in the holes;

S63, adding deionized water to H1N1 influenza virus standard antigen (batch number 19/312, purchased from NIBSC), dissolving the antigen to make the hemagglutinin content in the dissolved solution 40 μg/mL, and standing at room temperature for more than 5 minutes to obtain solution B;

S64, take the solution B obtained in step S63 and 10wt% 3-14 Detergent was mixed at a volume ratio of 9:1 and placed at 25°C for 30 minutes for lysis to obtain lysed solution C; the lysed solution C was diluted with 0.02 mol/L PBS buffer by 3/4, 2/4, and 1/4 times to obtain lysed solution C1, lysed solution C2, and lysed solution C3 respectively; the reconstituted H1N1 influenza virus antigen reference obtained in step S61 was mixed with 10 wt%/> 3-14 Detergent was mixed at a volume ratio of 9:1 and placed at 25°C for 30 minutes for lysis to obtain a lysed H1N1 influenza virus antigen reference substance;

S65. Use an adjustable range pipette to add the lysed solution C, lysed solution C1, lysed solution C2, lysed solution C3 obtained in step S64, and the lysed H1N1 influenza virus antigen reference obtained in step S64 to the agar plate wells, adding 10 μL to each well. After the liquid in the well is completely absorbed, place the gel plate in a wet box and let it stand and diffuse at 20-25° C. for 18-24 hours.

S66. After taking out the agar plate, soak it in 0.02 mol/L PBS buffer for 1 h to remove the serum in the gel;

S67, place the agar plate on a glass plate, cover it with 15 layers of filter paper, and finally press a 2kg marble plate on it for 1 hour until the agar plate becomes a thin layer;

S68. Place the thinned agar plate in a 50°C drying oven until completely dry;

S69, staining with a staining solution for 5 minutes until the agar plate turns blue, taking it out and rinsing it with deionized water to remove the surface staining solution, decolorizing it with a decolorizing solution until the precipitation ring is clear and the background turns light blue or transparent, and rinsing it with deionized water;

S70, use a scanner to scan the image and then use the Immulab software to automatically measure the diameter of the precipitation ring;

S71, calculate a linear regression equation for the corresponding hemagglutinin content of the precipitation ring diameters formed by the lysed solution C, the lysed solution C1, the lysed solution C2, and the lysed solution C3 obtained in step S64, substitute the precipitation ring diameter formed by the lysed H1N1 influenza virus antigen reference substance into the linear regression equation, and the hemagglutinin content of the H1N1 influenza virus antigen reference substance can be obtained, and the calculation formula is as follows:

y＝ax+b

Formula (4)

Where y is the average diameter of the precipitation ring;

x is the hemagglutinin content.

According to 20 test results independently obtained from 20 batches, the average value is calculated as the provisional target value, and this provisional target value is used as the target value for the next month for quality control. After the end of one month, the test results of that month are combined with the previous 20 test results to calculate the cumulative average value (first month), and this cumulative average value is used as the target value for the next month; repeat the above operation process for 5 consecutive months, and use the cumulative average value calculated from the initial 20 test results and all the data collected in 5 months as the hemagglutinin standard value of the H1N1 influenza virus antigen reference product, that is, the hemagglutinin standard value of the H1N1 influenza virus antigen reference product ( _C0 is 27.68μg/mL).

The calibration steps for the hemagglutination titer of the H1N1 influenza virus antigen reference material are as follows:

S71, taking the H1N1 influenza virus antigen reference substance prepared in step S5, and reconstituted it with 0.5 mL of sterile water for injection to obtain the reconstituted H1N1 influenza virus antigen reference substance;

S72, adding 50 μL of sterile 0.9 wt % sodium chloride solution to each well of the blood coagulation plate;

S73. Add 50 μL of the reconstituted H1N1 influenza virus antigen reference to the first well of the hemagglutination plate, take out 50 μL from the first well and add it to the second well, mix by pipetting, and make 2-fold serial dilutions in sequence until a negative result appears in the red blood cell agglutination (100% of the red blood cells are not agglutinated), and discard 50 μL of the last well after mixing;

S74, add 50 μL of 1v% chicken red blood cell suspension to each well from low concentration to high concentration, tap the blood coagulation plate to fully mix the red blood cells and the H1N1 influenza virus antigen reference substance, and let it stand;

S75, take one well and add 50 μL of sterile 0.9 wt% sodium chloride solution, then add 50 μL of 1v% chicken red blood cell suspension. This control group can exclude the non-specific agglutination of red blood cells themselves;

S76, incubation: 25°C for 30 min;

S77. Observe the hemagglutination results of red blood cells according to the contents of Table 1, take the previous positive result before the negative result (100% non-agglutination of red blood cells) as the determination end point, and calculate the hemagglutination titer value of the H1N1 influenza virus antigen reference substance according to the contents of Table 2;

Based on 20 test results independently obtained from 20 batches, the average value is calculated as the provisional target value, and this provisional target value is used as the target value for the next month for quality control. After the end of one month, the test results of that month are combined with the previous 20 test results to calculate the cumulative average value (first month), and this cumulative average value is used as the target value for the next month; repeat the above operation process for 5 consecutive months, and use the cumulative average value calculated from the initial 20 test results and all the data collected in 5 months as the standard value of the hemagglutination titer of the H1N1 influenza virus antigen reference product, that is, the _T0 of the H1N1 influenza virus antigen reference product is 1:515.

Preparation Example 2 provides an H3N2 influenza virus antigen reference material, and its preparation steps are different from those of Preparation Example 1 in that the H1N1 influenza virus split vaccine monovalent stock solution is replaced by the H3N2 influenza virus split vaccine monovalent stock solution, the H1N1 influenza virus standard antiserum is replaced by the H3N2 influenza virus standard antiserum (batch number 19/316, purchased from NIBSC), and the H1N1 influenza virus standard antigen is replaced by the H3N2 influenza virus standard antigen (batch number 20/108, purchased from NIBSC), and the different steps are as follows:

S2. Take the monovalent stock solution of H3N2 influenza virus split vaccine (obtained from the company's production workshop) and dilute it with 0.02 mol/L PBS buffer to a dilution solution A with a hemagglutinin content of 36 μg/mL;

S3, taking 200 mL of the diluent A obtained in step S2, adding the lyophilization protective agent obtained in step S1, and obtaining a reference solution with a hemagglutinin content of 24 μg/mL;

S62. Add the H3N2 influenza virus standard antiserum to 25 mL of 1.5 wt% agarose solution at 56°C, shake it manually (shake it gently to avoid generating a large number of bubbles), and prepare a solution A containing 25 μL/mL of the H3N2 influenza virus standard antiserum; pour the agarose on a gel plate, and quickly remove any bubbles with a pipette; let it stand until the agarose is completely solidified, and then put it in a 5°C refrigerator for 20 min. Take out the refrigerated agar plate and use a hole puncher to punch holes with a hole diameter of 3 mm and a distance between holes of 1.5 cm. Punch 36 holes in a 10 cm×10 cm agar plate, and use a vacuum pump to carefully suck out the gel in the holes;

The hemagglutinin standard value of the H3N2 influenza virus antigen reference material (C ₀ is 24.48 μg/mL);

The standard value of the hemagglutination titer of the H3N2 influenza virus antigen reference substance (T ₀ is 1:503).

Preparation Example 3 provides a BV influenza virus antigen reference material, and its preparation steps are different from those of Preparation Example 1 in that the H1N1 influenza virus split vaccine monovalent stock solution is replaced by the BV influenza virus split vaccine monovalent stock solution, the H1N1 influenza virus standard antiserum is replaced by the BV influenza virus standard antiserum (batch number 19/218, purchased from NIBSC), and the H1N1 influenza virus standard antigen is replaced by the BV influenza virus standard antigen (batch number 19/208, purchased from NIBSC), and the different steps are as follows:

S2. Take the monovalent stock solution of influenza virus split vaccine type BV (obtained from the production workshop of our company) and dilute it with 0.02 mol/L PBS buffer to dilute the solution A with a hemagglutinin content of 42 μg/mL;

S3, taking 200 mL of the dilution solution A obtained in step S2, adding the lyophilization protective agent obtained in step S1, and obtaining a reference solution with a hemagglutinin content of 28 μg/mL;

The hemagglutinin standard value of the BV influenza virus antigen reference substance (C ₀ is 28.53 μg/mL);

The standard value of the hemagglutination titer of the BV influenza virus antigen reference substance (T ₀ is 1:1492).

Preparation Example 4 provides a BY influenza virus antigen reference material, and its preparation steps are different from those of Preparation Example 1 in that the H1N1 influenza virus split vaccine monovalent stock solution is replaced by the BY influenza virus split vaccine monovalent stock solution, the H1N1 influenza virus standard antiserum is replaced by the BY influenza virus standard antiserum (batch number 17/214, purchased from NIBSC), and the H1N1 influenza virus standard antigen is replaced by the BY influenza virus standard antigen (batch number 16/158, purchased from NIBSC), and the different steps are as follows:

S2. Take the BY influenza virus split vaccine monovalent stock solution (obtained from the company's production workshop) and dilute it with 0.02 mol/L PBS buffer to a diluent A with a hemagglutinin content of 36 μg/mL;

S3, taking 200 mL of the dilution solution A obtained in step S2, adding the lyophilization protective agent obtained in step S1, and obtaining a reference solution with a hemagglutinin content of 24 μg/mL;

S62. Add BY influenza virus standard antiserum to 25 mL of 1.5 wt% agarose solution at 56°C, shake by hand (shake gently to avoid generating a large number of bubbles), and prepare solution A with a BY influenza virus standard antiserum content of 45 μL/mL; pour agarose on a gel plate, and quickly remove bubbles with a pipette if any; let stand until the agarose is completely solidified, place in a 5°C refrigerator for 20 min, take out the refrigerated agar plate, and use a hole puncher to punch holes with a hole diameter of 3 mm and a hole distance of 1.5 cm. Punch 36 holes in a 10 cm×10 cm agar plate, and use a vacuum pump to carefully suck out the gel in the holes;

The hemagglutinin standard value of the BY influenza virus antigen reference substance (C ₀ is 23.39 μg/mL);

The standard value of the hemagglutination titer of the BY type influenza virus antigen reference substance (T ₀ is 1:786).

Example

Embodiments 1-4 provide a method for detecting hemagglutinin in a monovalent stock solution of an influenza virus split vaccine, which is described below using Embodiment 1 as an example.

The method for detecting hemagglutinin of the monovalent stock solution of influenza virus split vaccine provided in Example 1 comprises the following steps:

S1. Cracking treatment

The H1N1 influenza virus split vaccine monovalent stock solution was diluted 15 times with 0.02 mol/L PBS buffer to obtain a H1N1 influenza virus split vaccine monovalent stock solution dilution solution;

Take the H1N1 influenza virus antigen reference substance and reconstitute it with 0.5 mL of sterile water for injection to obtain the H1N1 influenza virus antigen reference substance solution;

10 wt % of the H1N1 influenza virus split vaccine monovalent stock solution dilution and the H1N1 influenza virus antigen reference solution were added respectively. 3-14 Detergent (H1N1 influenza virus split vaccine monovalent stock solution dilution and 10wt%/> The volume ratio of 3-14 Detergent is 9:1, H1N1 influenza virus antigen reference solution and 10wt%/> The volume ratio of 3-14 Detergent was 9:1), and the virus was lysed for 30 minutes to obtain a lysed H1N1 influenza virus lysed vaccine monovalent stock solution dilution and a lysed H1N1 influenza virus antigen reference solution.

S2. Hemagglutination titer test

S21, blood coagulation plate marking: add 50 μL of sterile 0.9 wt % sodium chloride solution to each well of the blood coagulation plate;

S22, serial dilution: add 50 μL of the split H1N1 influenza virus split vaccine monovalent stock solution dilution solution and the split H1N1 influenza virus antigen reference solution to the first well of the blood agglutination plate, take out 50 μL from the first well and add it to the second well, mix by pipetting, and make 2-fold serial dilutions in sequence until the red blood cell agglutination shows a negative result (100% of the red blood cells are not agglutinated), and discard 50 μL of the last well after mixing;

S23, adding chicken red blood cell suspension: adding 50 μL 1wt% chicken red blood cell suspension to each well from low concentration to high concentration, tapping the hemagglutination plate to fully mix the red blood cells with the split H1N1 influenza virus split vaccine monovalent stock solution dilution and the split H1N1 influenza virus antigen hemagglutination titer reference solution, and then letting it stand;

S24, negative control to exclude non-specific agglutination of erythrocytes themselves: take one well and first add 50 μL of sterilized 0.9% sodium chloride solution, then add 50 μL of 1wt% chicken blood erythrocyte suspension. This group of controls can exclude non-specific agglutination of erythrocytes themselves;

S25, incubation: 25°C for 30 min;

S26. Calculate the hemagglutination titer value according to the red blood cell agglutination result: According to the contents of Table 1 and Table 2, the previous positive result before the negative result (100% non-agglutination of red blood cells) is used as the endpoint to determine the dilution multiple of the previous positive result before the negative result occurs. According to the hemagglutination titer calculation method in Table 2, calculate the hemagglutination titer value T _t of the H1N1 influenza virus split vaccine monovalent stock solution dilution and the hemagglutination titer value T _s of the H1N1 influenza virus antigen reference substance.

S3. Calculate the standard value of the hemagglutination titer of the monovalent stock solution of the H1N1 influenza virus split vaccine. Take the ratio between the hemagglutination titer detection value _Tt of the diluent of the monovalent stock solution of the H1N1 influenza virus split vaccine and the hemagglutination titer detection value _Ts of the H1N1 influenza virus antigen reference as the correction coefficient, correct the hemagglutination titer detection value of the diluent of the monovalent stock solution of the H1N1 influenza virus split vaccine, and obtain the standard value of the hemagglutination titer of the monovalent stock solution of the H1N1 influenza virus split vaccine. The calculation formula is shown in Formula 3:

Wherein, T is the standard value of the hemagglutination titer of the monovalent stock solution of the H1N1 influenza virus split vaccine;

_Tt is the hemagglutination titer test value of the monovalent stock solution dilution of H1N1 influenza virus split vaccine;

_Ts is the hemagglutination titer test value of the H1N1 influenza virus antigen reference substance;

T ₀ is the standard value of the hemagglutination titer of the H1N1 influenza virus antigen reference substance (T ₀ =1:515);

X is the dilution multiple of the H1N1 influenza virus split vaccine monovalent stock solution dilution (the value of X is 15).

Note: Each sample was tested 6 times in parallel. The test results of the hemagglutination titer of the monovalent stock solution of H1N1 influenza virus split vaccine are shown in Table 3.

Table 3 Test results of hemagglutination titer of monovalent stock solution of H1N1 influenza virus split vaccine

S4. Calculation of the hemagglutinin standard value of the monovalent stock solution of the H1N1 influenza virus split vaccine The hemagglutinin standard value of the monovalent stock solution of the H1N1 influenza virus split vaccine is calculated from the hemagglutination titer standard value of the monovalent stock solution of the H1N1 influenza virus split vaccine, and the calculation formula is shown in Formula 2:

Wherein, C is the hemagglutinin standard value of the monovalent stock solution of H1N1 influenza virus split vaccine;

T is the standard value of the hemagglutination titer of the monovalent stock solution of the H1N1 influenza virus split vaccine;

T ₀ is the standard value of the hemagglutination titer of the H1N1 influenza virus antigen reference substance (T ₀ =1:515);

C ₀ is the standard value of hemagglutinin of the H1N1 influenza virus antigen reference substance (C ₀ = 27.68 μg/mL).

The test results of hemagglutinin of the monovalent stock solution of H1N1 influenza virus split vaccine are shown in Table 4.

Table 4 Test results of hemagglutinin in monovalent stock solution of H1N1 influenza virus split vaccine

As shown in Table 4, the detection method of hemagglutinin in the monovalent stock solution of H1N1 influenza virus split vaccine has good precision, good repeatability and good stability, and can be used for the quality control evaluation of the monovalent stock solution of H1N1 influenza virus split vaccine.

Example 2 is different from Example 1 in that an equal volume of the H1N1 influenza virus split vaccine monovalent stock solution is replaced by an H3N2 influenza virus split vaccine monovalent stock solution, the dilution multiple of the H3N2 influenza virus split vaccine monovalent stock solution is 10 times, the test results of the hemagglutination titer of the H3N2 influenza virus split vaccine monovalent stock solution are shown in Table 5 (wherein, the hemagglutination titer standard value T ₀ of the H3N2 influenza virus antigen reference substance is 1:503), and the test results of the hemagglutinin of the H3N2 influenza virus split vaccine monovalent stock solution are shown in Table 6 (wherein, the hemagglutinin standard value C ₀ of the H3N2 influenza virus antigen reference substance is 24.48 μg/mL).

Table 5 Test results of hemagglutination titer of monovalent stock solution of H3N2 influenza virus split vaccine

Table 6 Test results of hemagglutinin in monovalent stock solution of H3N2 influenza virus split vaccine

As shown in Table 6, the detection method of hemagglutinin in the monovalent stock solution of H3N2 influenza virus split vaccine has good precision, good repeatability and good stability, and can be used for the quality control evaluation of the monovalent stock solution of H3N2 influenza virus split vaccine.

Example 3 is different from Example 1 in that an equal volume of the H1N1 influenza virus split vaccine monovalent stock solution is replaced by a BV influenza virus split vaccine monovalent stock solution, the dilution multiple of the BV influenza virus split vaccine monovalent stock solution is 6, the test results of the hemagglutination titer of the BV influenza virus split vaccine monovalent stock solution are shown in Table 7 (wherein, the hemagglutination titer standard value T ₀ of the BV influenza virus antigen reference substance is 1:1492), and the test results of the hemagglutinin of the BV influenza virus split vaccine monovalent stock solution are shown in Table 8 (wherein, the hemagglutinin standard value C ₀ of the BV influenza virus antigen reference substance is 28.53 μg/mL).

Table 7 Test results of hemagglutination titer of BV influenza virus split vaccine monovalent stock solution

Table 8 Test results of hemagglutinin in monovalent stock solution of influenza virus split vaccine type BV

As shown in Table 8, the detection method of hemagglutinin in the monovalent stock solution of BV influenza virus split vaccine has good precision, good repeatability and good stability, and can be used for the quality control evaluation of the monovalent stock solution of BV influenza virus split vaccine.

Example 4 is different from Example 1 in that an equal volume of the H1N1 influenza virus split vaccine monovalent stock solution is replaced by a BY influenza virus split vaccine monovalent stock solution, the dilution multiple of the BY influenza virus split vaccine monovalent stock solution is 10, the test results of the hemagglutination titer of the BY influenza virus split vaccine monovalent stock solution are shown in Table 9 (wherein, the standard value of the hemagglutination titer of the BY influenza virus antigen reference substance T ₀ =1:786), and the test results of the hemagglutinin of the BY influenza virus split vaccine monovalent stock solution are shown in Table 10 (wherein, the standard value of the hemagglutinin of the BY influenza virus antigen reference substance C ₀ =23.39 μg/mL).

Table 9 Test results of hemagglutination titer of BY influenza virus split vaccine monovalent stock solution

Table 10 Test results of hemagglutinin in monovalent stock solution of BY influenza virus split vaccine

As shown in Table 10, the detection method of hemagglutinin in BY-type influenza virus split vaccine monovalent stock solution has good precision, good repeatability and good stability, and can be used for the quality control evaluation of BY-type influenza virus split vaccine monovalent stock solution.

Accuracy analysis of hemagglutinin detection method

The hemagglutinin of the hemagglutination titer reference substance of influenza virus antigen with different concentrations was measured, and the recovery rate was calculated (recovery rate = measured value/true value×100%). The accuracy of the hemagglutinin detection method was analyzed based on the recovery rate.

Example 6 is different from Example 1 in that an equal volume of the H1N1 influenza virus monovalent stock solution is replaced by the H1N1 influenza virus antigen reference substance prepared in Preparation Example 1. The hemagglutination titer, hemagglutinin and hemagglutinin recovery rate of the H1N1 influenza virus antigen reference substance are shown in Table 11.

Table 11 Hemagglutination titer, hemagglutinin and hemagglutinin recovery of H1N1 influenza virus antigen reference

Example 7 is different from Example 6 in that the concentration of the H1N1 influenza virus antigen reference substance prepared in Preparation Example 1 is replaced from 100% to 80%. The hemagglutination titer, hemagglutinin and hemagglutinin recovery rate of the 80% concentration H1N1 influenza virus antigen reference substance are shown in Table 12.

Table 12 Hemagglutination titer, hemagglutinin and hemagglutinin recovery of 80% concentration H1N1 influenza virus antigen reference material

It can be seen from Tables 11-12 that the method for detecting hemagglutinin in the monovalent stock solution of influenza virus split vaccine provided in the present application can accurately analyze the hemagglutinin content of influenza virus antigen samples with high accuracy.

Comparative Example

Comparative Example 1 provides a conventional one-way immunodiffusion method for testing the hemagglutinin of the monovalent stock solution of influenza virus split vaccine, and the detection steps are as follows:

S1. Preparation and punching of agarose: Add H1N1 influenza virus standard antiserum (batch number 19/314, purchased from NIBSC) to 25 mL of 1.5 wt% agarose solution at 56°C, shake by hand (shake gently to avoid generating a large number of bubbles), and prepare solution A with a content of 35 μL/mL of H1N1 influenza virus standard antiserum; pour agarose on the gel plate, and quickly remove bubbles with a pipette if any; let stand until the agarose is completely solidified, put it in a 5°C refrigerator for 20 min, take out the refrigerated agar plate, and use a hole puncher to punch holes with a hole diameter of 3 mm and a distance between holes of 1.5 cm. Punch 36 holes in a 10 cm×10 cm agar plate, and carefully suck out the gel in the holes with a vacuum pump;

S2. Preparation of H1N1 influenza virus standard antigen solution: Add deionized water to H1N1 influenza virus standard antigen (batch number 19/312, purchased from NIBSC) to dissolve the solution until the hemagglutinin content in the solution is 40 μg/mL, and let stand at room temperature for more than 5 minutes to obtain solution B;

S3, H1N1 influenza virus standard antigen solution and H1N1 influenza virus split vaccine monovalent stock solution lysis and dilution: take the solution B obtained in step S2 and 10wt% 3-14 Detergent was mixed at a volume ratio of 9:1 and placed at 25°C for 30 minutes for lysis to obtain a lysed solution C; the lysed solution C was diluted 3/4, 2/4, and 1/4 times with 0.02mol/L PBS buffer to obtain lysed solution C1, lysed solution C2, and lysed solution C3 respectively; the H1N1 influenza virus split vaccine monovalent stock solution was diluted 15 times with 0.02mol/L PBS buffer to obtain a H1N1 influenza virus split vaccine monovalent stock solution dilution, and the H1N1 influenza virus split vaccine monovalent stock solution dilution was then mixed with 10wt% 3-14 Detergent was mixed at a volume ratio of 9:1 and placed at 25°C for 30 minutes for lysis to obtain a monovalent stock solution of H1N1 influenza virus lysed vaccine;

S4. Sample loading and diffusion: Use an adjustable range pipette to add the lysed solution C, lysed solution C1, lysed solution C2, lysed solution C3 obtained in step S3, and the lysed H1N1 influenza virus split vaccine monovalent stock solution obtained in step S3 to the agar plate wells, adding 10 μL to each well. After the liquid in the well is completely absorbed, place the gel plate in a wet box and let it stand at 20°C for 24 hours for diffusion;

S5, gel washing: After taking out the agar plate, soak it in 0.02 mol/L PBS buffer for 1 h to remove the serum in the gel;

S6, glue pressing: Place the agar plate on a glass plate, cover it with 15 layers of filter paper, and finally press a 2kg marble plate on it for 1 hour until the agar plate becomes a thin layer;

S7, drying: put the thinned agar plate into a 50°C drying oven until it is completely dry;

S8, staining and decolorization: stain with staining solution for 5 minutes until the agar plate turns blue, take it out and rinse with deionized water to remove the surface staining solution, decolorize with decolorizing solution until the precipitation ring is clear and the background turns light blue or transparent, and rinse with deionized water;

S9, Determination of precipitation ring diameter: Scan the image with a scanner and then use Immulab software for automatic measurement;

S10, obtain a linear regression equation for the diameters of the precipitation rings formed by the lysed solutions C, C1, C2, and C3 obtained in step S64 and their corresponding hemagglutinin contents, substitute the diameters of the precipitation rings formed by the lysed H1N1 influenza virus split vaccine monovalent stock solution into the linear regression equation to obtain x (hemagglutinin content), the linear regression equation is formula (4), and then multiply x by 15 (dilution multiple) to obtain the hemagglutinin content of the H1N1 influenza virus split vaccine monovalent stock solution.

The hemagglutinin content of the monovalent stock solution of the H1N1 influenza virus split vaccine measured in Comparative Example 1 is shown in Table 13.

Table 13 Test results of hemagglutinin in comparative example 1

By comparing the test results of Table 4 and Table 13, it can be seen that the hemagglutinin test results determined by the hemagglutinin detection method for the monovalent stock solution of influenza virus split vaccine provided in the present application are stable, and the hemagglutinin content obtained from different test data is stable, especially the six hemagglutinin test results of the monovalent stock solution of H1N1 influenza virus split vaccine with batch numbers A1-1 and A1-2 are consistent; while the six hemagglutinin test results of the monovalent stock solution of H1N1 influenza virus split vaccine corresponding to the conventional unidirectional immunodiffusion method are all different, indicating that the hemagglutinin detection method for the monovalent stock solution of influenza virus split vaccine provided in the present application is more stable and accurate than the conventional unidirectional immunodiffusion method. This is because the present application avoids the influence of factors such as agarose concentration, toughness of the gel plate, staining time, decolorization time, shape and depth of the precipitation ring on the hemagglutinin detection results, thereby improving the stability, accuracy and sensitivity of the detection results.

This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims (10)

1. The method for detecting the monovalent stock solution hemagglutinin of the influenza virus split vaccine is characterized by comprising the following steps of:

s1, cracking treatment: respectively adding a cracking agent into the influenza virus cracked vaccine monovalent stock solution and the influenza virus antigen reference for cracking to obtain cracked influenza virus cracked vaccine monovalent stock solution and cracked influenza virus antigen reference; the preparation method of the influenza virus antigen reference comprises the following steps:

Diluting an influenza virus split vaccine monovalent stock solution to a diluent with the hemagglutinin content of 42ug/mL by using a buffer solution, adding a freeze-drying protective agent to obtain a reference solution with the hemagglutinin content of 28ug/mL, and freeze-drying the obtained reference solution to obtain an influenza virus antigen reference;

S2, detecting the blood coagulation titer: detecting the hemagglutination titer of the split influenza virus split vaccine monovalent stock solution and the hemagglutination titer of the split influenza virus antigen reference respectively to obtain a hemagglutination titer detection value of the split influenza virus split vaccine monovalent stock solution and a hemagglutination titer detection value of the split influenza virus antigen reference;

s3, calculating a standard value of the hemagglutination titer of the monovalent stock solution of the influenza virus split vaccine: calculating the standard value of the hemagglutination titer of the monovalent stock solution of the influenza virus split vaccine according to the detection value of the hemagglutination titer of the monovalent stock solution of the influenza virus split vaccine, wherein the calculation formula is shown in formula 1:

wherein T is the standard value of the hemagglutination titer of the monovalent stock solution of the influenza virus split vaccine;

T _t is the hemagglutination titer detection value of the influenza virus split vaccine monovalent stock solution;

T _s is the hemagglutination titer detection value of the influenza virus antigen reference;

T ₀ is the standard value of the hemagglutination titer of the influenza virus antigen reference;

S4, calculating a hemagglutinin standard value of the influenza virus split vaccine monovalent stock solution: calculating the standard value of hemagglutinin of the monovalent stock solution of the influenza virus split vaccine according to the standard value of hemagglutinin of the monovalent stock solution of the influenza virus split vaccine, wherein the calculation formula is shown in formula 2:

Wherein C is the standard value of hemagglutinin of the monovalent stock solution of the influenza virus split vaccine;

T is the standard value of the hemagglutination titer of the monovalent stock solution of the influenza virus split vaccine;

T ₀ is the standard value of the hemagglutination titer of the influenza virus antigen reference;

C ₀ is the standard value of hemagglutinin of influenza virus antigen reference.

2. The method for detecting hemagglutinin of an influenza virus split vaccine monovalent stock solution according to claim 1, wherein the influenza virus split vaccine monovalent stock solution is any one of an H1N1 type influenza virus monovalent stock solution, an H3N2 type influenza virus monovalent stock solution, a BV type influenza virus monovalent stock solution or a BY type influenza virus monovalent stock solution.

3. The method for detecting hemagglutinin of monovalent stock solution of influenza virus split vaccine according to claim 1, wherein in the step S1, the monovalent stock solution of influenza virus split vaccine needs to be diluted with PBS buffer solution until the antigen content of the influenza virus split vaccine is 10-50 μg/mL before adding the splitting agent, so as to obtain a monovalent stock solution dilution of influenza virus split vaccine; the influenza virus antigen reference is re-dissolved by sterilizing water for injection before adding the lysing agent, so as to obtain an influenza virus antigen reference solution.

4. The method for detecting hemagglutinin of monovalent stock solution of influenza virus split vaccine according to claim 3, wherein in the step S1, the volume ratio of the monovalent stock solution of influenza virus split vaccine to the splitting agent and the volume ratio of the influenza virus antigen reference solution to the splitting agent are (8-10): 1.

5. The method for detecting the monovalent stock solution hemagglutinin of the influenza virus split vaccine according to claim 4, wherein in the step S1, the time of the splitting treatment is 20-40 min.

6. The method for detecting hemagglutinin of monovalent stock solution of influenza virus split vaccine according to claim 4, wherein said splitting agent is selected from the group consisting of Triton X-100, polysorbate 80, triton N101 and combinations thereof3-14 Detergent.

7. The method for detecting influenza virus split vaccine monovalent stock hemagglutinin according to claim 6, wherein said method comprises the steps ofThe concentration of 3-14Detergent was 10wt%.

8. The method for detecting hemagglutinin of monovalent stock solution of influenza virus split vaccine according to claim 7, wherein in said step S1, said monovalent stock solution of influenza virus split vaccine is diluted with3-14Detergent volume ratio, influenza Virus antigen reference solution and/>The volume ratio of 3-14Detergent is 9:1.

9. The method for detecting hemagglutinin of monovalent stock solution of split influenza virus vaccine according to claim 1, wherein in the step S2, the specific step of detecting the hemagglutination titer comprises:

S21, labeling the blood coagulation plate;

s22, serial dilution;

s23, adding chicken erythrocyte suspension;

s24, negative control eliminates nonspecific agglutination of erythrocytes themselves;

s25, incubating;

s26, calculating a hemagglutination titer detection value according to the hemagglutination result.

10. The method for detecting the monovalent stock solution hemagglutinin of the split influenza virus vaccine according to claim 9, wherein the operation step of S26 comprises:

And observing the hemagglutination result, and respectively calculating the hemagglutination titer detection value of the monovalent stock solution of the influenza virus split vaccine and the hemagglutination titer detection value of the influenza virus antigen reference by taking the previous positive with the negative result as a judging end point.