A method for determining the dissociation solution and antigen content of influenza vaccines adsorbed with aluminum adjuvant.

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a dissociation solution for aluminum adjuvant-adsorbed influenza vaccines and a method for determining antigen content. The dissociation solution of this invention is composed of potassium phosphate solution, diethanolamine, Triton X-100, Tween-20, dodecyl dimethyl betaine, and water. This dissociation solution exhibits excellent dissociation efficiency, achieving a recovery rate of 106%-110% for hemagglutinin from H1N1, H3N2, and BV influenza vaccines. It effectively protects antigen activity and is simple to operate with wide applicability. The dissociated sample can be directly used for mainstream detection methods such as one-way immunodiffusion assay, enzyme-linked immunosorbent assay, and high-performance liquid chromatography, enabling precise quantification of antigen content in aluminum adjuvant-adsorbed influenza vaccines and providing reliable technical support for vaccine quality control.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for determining the dissociation solution and antigen content of an aluminum adjuvant-adsorbed influenza vaccine. Background Technology

[0002] Currently, the gold standard method for evaluating influenza vaccine batches is the single-path immune diffusion (SRID) assay. The principle of this assay is to first mix a certain amount of antibody in an agarose gel, allowing the antigen solution to diffuse freely from a localized area outwards within the agarose gel, forming a visible precipitation ring within a specific region. The diameter or area of ​​the ring is positively correlated with the antigen content. Applying this method to influenza vaccines, by comparing the precipitation ring produced by an unknown sample with the precipitation ring of a standard with known hemagglutinin (HA) content, the HA content of the unknown sample can be determined.

[0003] Aluminum adjuvants are the most widely used human vaccine adjuvants to date, effectively enhancing the immunogenicity of vaccines. Studies show that the possible mechanisms of action of aluminum adjuvants include: antigen storage; enhancing the body's innate immune response; enhancing antigen presentation; enhancing Th2 cell-mediated adaptive immune responses; activating B cells to induce antibody production; and activating complement function.

[0004] Accurately evaluating the hemagglutinin content in influenza vaccines is crucial, as it is an important indicator for ensuring vaccine safety and efficacy. However, hemagglutinin adsorbed onto aluminum cannot be readily detected directly using one-way immunodiffusion. First, the interaction between hemagglutinin and the aluminum adjuvant needs to be broken, allowing the hemagglutinin to dissociate from the aluminum adjuvant, before quantitative detection is performed using one-way immunodiffusion.

[0005] According to literature reports, inorganic bases, organic acids, some metal ion chelates, surfactants, and other substances can specifically disrupt the interaction between antigens and adjuvants, achieving the purpose of antigen dissociation. It is necessary to develop a novel dissociation solution for aluminum-adsorbed influenza vaccines and to improve the SRID assay for use in aluminum-adsorbed influenza vaccines. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a method for determining the dissociation solution and antigen content of an aluminum adjuvant-adsorbed influenza vaccine.

[0007] the term:

[0008] The term "aluminum adjuvant adsorbed influenza vaccine" as used in this invention refers to an influenza vaccine using aluminum hydroxide as an adjuvant. The aluminum adjuvant adsorbs the core antigens (such as hemagglutinin) in the vaccine, enhancing the immune effect, but the antigen content can only be detected after dissociation.

[0009] The term "dissociation solution" as used in this invention refers to a solution specifically designed to break the adsorption between the aluminum adjuvant and the antigen, thereby releasing the antigen from the aluminum adjuvant for subsequent detection.

[0010] The term "hemagglutinin" used in this invention refers to the core antigen protein in influenza vaccines. Its content is a key indicator for evaluating vaccine effectiveness and is also the target substance for dissociation and detection in this invention.

[0011] The term "specific antiserum" used in this invention refers to specific antibodies (rabbit-derived) against H1N1, H3N2, and BV influenza antigens. These antibodies form a visible precipitate ring upon binding to the corresponding antigens and are used for quantitative detection.

[0012] The term "Single-directional immune diffusion assay (SRID)" used in this invention refers to the core detection method of this invention, in which antibodies are mixed into agar gel, and antigens diffuse from the sample wells to the surrounding area. The diameter of the precipitate ring formed is positively correlated with the antigen content. The antigen concentration in the sample is calculated through a standard curve, which is the gold standard for quantifying influenza vaccine antigens.

[0013] The term "bidirectional immunodiffusion assay" used in this invention refers to the diffusion of antibodies and antigens from different wells of a gel, forming a precipitation line at an appropriate concentration, which is used for antigen specificity verification or qualitative detection.

[0014] The term "enzyme-linked immunosorbent assay (ELISA)" used in this invention refers to the quantitative detection of antigen content through the specific binding of antigen and antibody, combined with the color development of enzyme markers.

[0015] The term “SDS-polyacrylamide gel electrophoresis (SDS-PAGE)” as used in this invention refers to an electrophoretic technique for separating proteins by molecular weight differences to separate antigens from other proteins for purity analysis.

[0016] The term "Western blot detection" used in this invention refers to the use of specific antibody hybridization and color development based on SDS-PAGE to verify the presence of the target antigen, thus combining separation and identification functions.

[0017] The term "high performance liquid chromatography (HPLC)" as used in this invention refers to the rapid and highly accurate separation of sample components by means of a chromatographic column, and the quantification of antigens based on their retention time and peak area.

[0018] The term "influencing factor sample" as used in this invention refers to a sample of trivalent recombinant influenza protein vaccine (Sf9 cells) placed at 37°C.

[0019] The technical solution of this invention is as follows:

[0020] On one hand, the present invention provides a dissociation solution for adsorbing influenza vaccines with aluminum adjuvants, the dissociation solution being composed of potassium phosphate solution, diethanolamine, Triton X-100, Tween-20, dodecyl dimethyl betaine and water.

[0021] Specifically, the dissociation solution consists of 0.4 mol / L-0.5 mol / L potassium phosphate solution, 2%-3% v / v diethanolamine, 0.2%-0.6% v / v Triton X-100, 0.1%-0.4% v / v Tween-20, 0.4%-0.8% w / w dodecyl dimethyl betaine, and water.

[0022] Preferably, the dissociation solution consists of a 0.5 mol / L potassium phosphate solution, 2.5% v / v diethanolamine, 0.4% v / v Triton X-100, 0.2% v / v Tween-20, 0.4% w / w dodecyl dimethyl betaine, and water.

[0023] On the other hand, the present invention provides a method for preparing the dissociation solution according to any one of the above claims, the method comprising: mixing potassium phosphate solution, diethanolamine, Triton X-100, Tween-20, dodecyl dimethyl betaine and water evenly.

[0024] Specifically, the preparation method includes preparing a mother liquor:

[0025] (1) 1M potassium phosphate solution: Weigh 18.26g of dipotassium hydrogen phosphate trihydrate and 2.72g of potassium dihydrogen phosphate, and dissolve them in 100ml of water.

[0026] (2) 20% v / v diethanolamine: Dissolve 2 ml of diethanolamine in 8 ml of water.

[0027] (3) 10% v / v TritonX-100: Dissolve 5 ml of TritonX-100 in 45 ml of water.

[0028] (4) 10% v / v Tween-20: Dissolve 1 ml of Tween-20 in 10 ml of water.

[0029] (5) 30% w / w dodecyl dimethyl betaine: purchased from Maclean, catalog number D848892.

[0030] Specifically, the preparation method further includes: thoroughly mixing water, 1M potassium phosphate solution, 20% v / v diethanolamine, 10% v / v Triton X-100, 10% v / v Tween-20, and 30% w / w dodecyl dimethyl betaine.

[0031] In another aspect, the present invention provides an antigen dissociation method for adsorbing influenza vaccines with aluminum adjuvants, wherein the antigen dissociation method includes using the dissociation solution or reagent described in any of the above-mentioned claims.

[0032] Specifically, the antigen dissociation method includes the following steps: adsorbing influenza vaccine with aluminum adjuvant, centrifuging, discarding the supernatant, adding dissociation solution to the precipitate, incubating, centrifuging, taking the supernatant, and obtaining the dissociated antigen.

[0033] Preferably, the volume ratio of the precipitate to the dissociation solution is 1:8-10.

[0034] More preferably, the volume ratio of the precipitate to the dissociation solution is 1:8-9 or 1:9-10.

[0035] More preferably, the volume ratio of the precipitate to the dissociation liquid is 1:9.

[0036] Preferably, the incubation includes placing the food at 25-37°C for 3-4 hours.

[0037] More preferably, the incubation includes placing the food at 25-26, 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36 or 36-37°C for 3-4 hours.

[0038] In another preferred embodiment, the incubation includes placing the food at 37°C for 3 hours.

[0039] Preferably, the centrifugation includes centrifugation at 8000-12000g for 5-10 minutes.

[0040] More preferably, the centrifugation includes centrifugation at 8000-9000, 9000-10000, 10000-11000 or 11000-12000g for 5-6, 6-7, 7-8, 8-9 or 9-10 minutes.

[0041] In another preferred embodiment, the centrifugation includes centrifugation at 10000 rpm for 5 minutes.

[0042] In another aspect, the present invention provides a method for detecting the antigen content in an aluminum adjuvant-adsorbed influenza vaccine. The detection method includes a pretreatment method for the aluminum adjuvant-adsorbed influenza vaccine, wherein the pretreatment method includes using the dissociation solution or reagent described in any of the above-mentioned methods to dissociate the antigen in the aluminum adjuvant-adsorbed influenza vaccine.

[0043] Specifically, the detection methods include, but are not limited to, any one or more of the following: one-way immunodiffusion assay, two-way immunodiffusion assay, enzyme-linked immunosorbent assay, hemagglutination inhibition assay, SDS-polyacrylamide gel electrophoresis, Western blot detection, and high performance liquid chromatography.

[0044] Preferably, the detection method is a one-way immunodiffusion assay or high-performance liquid chromatography.

[0045] The beneficial effects of this invention are as follows:

[0046] The dissociation solution provided by this invention has excellent dissociation efficiency and high specificity. It can effectively destroy the adsorption force between aluminum adjuvant and hemagglutinin. The hemagglutinin dissociation recovery rate of H1N1, H3N2 and BV influenza vaccines reaches 106%-110%, which is far superior to existing dissociation solutions. Moreover, it does not cross-react with other antigen types, and its specificity meets the strict requirements of vaccine testing.

[0047] The dissociation solution provided by this invention effectively protects antigen activity. The proportions of each component have been scientifically optimized to avoid hemagglutinin denaturation while achieving efficient dissociation, ensuring that the antigen can still stably bind to specific antibodies after dissociation. Combined with standardized dissociation procedures, the linear correlation coefficient R of the detection results is high. 2 With a strength of ≥0.95, the recovery rate, precision (RSD≤5.71%), and durability (stable within 72 hours) all meet the vaccine quality control standards, enabling precise quantification of hemagglutinin content.

[0048] This invention is simple, efficient, and widely adaptable. The dissociation process requires no special equipment, is simple in steps, and takes only 3 hours. The dissociated sample can be directly adapted to mainstream detection technologies such as one-way immunodiffusion assay, ELISA, and HPLC. It is especially compatible with SRID assay, requiring no additional optimization of the detection process, and is easy to apply on a large scale in laboratories. Attached Figure Description

[0049] Figure 1This is a graph showing the results of the specificity test; A in the graph represents the specificity test results for H1N1 serum. Wells 1-7 contain the self-made standard antigen of H1N1 serum (Well 1: 40 μg / mL, Well 2: 30 μg / mL, Well 3: 25 μg / mL, Well 4: 20 μg / mL, Well 5: 10 μg / mL, Well 6: 6 μg / mL, Well 7: 6 μg / mL), Well 8 contains the 30 μg / mL self-made standard antigen of H3N2, and Well 9 contains 30 μg / mL. BV type self-made standard antigen; B represents the H3N2 type serum specificity test results, wells 1-7 are the H3N2 type serum self-made standard antigen (well 1: 40 μg / mL, well 2: 30 μg / mL, well 3: 25 μg / mL, well 4: 20 μg / mL, well 5: 10 μg / mL, well 6: 6 μg / mL, well 7: 6 μg / mL), well 8 is the 30 μg / mL H1N1 type self-made standard antigen, and well 9 is the 30 μg / mL BV type self-made standard antigen; C represents the BV type serum specificity test results, wells 1-7 in the figure are the BV type serum self-made standard antigen (well 1: 40 μg / mL, well 2: 30 μg / mL, well 3: 25 μg / mL, well 4: 20 μg / mL, well 5: 10 μg / mL, well 6: 6 μg / mL, well 7: 6 μg / mL), and well 8 is 30 μg / mL. H1N1 type self-made standard antigen, well 9 contains 30 μg / mL H3N2 type self-made standard antigen.

[0050] Figure 2 To compare the effect with the dissociation solution in the prior art; wells 1-5 in the figure are standard curves (well 1: 40 μg / mL, well 2: 30 μg / mL, well 3: 25 μg / mL, well 4: 20 μg / mL, well 5: 10 μg / mL), well 6 is a positive control, well 7 is the dissociation result of the influencing factor sample C, well 8 is the result of dissociation solution C, well 9 is the dissociation result of the influencing factor sample A, well 10 is the result of dissociation solution A, well 11 is the dissociation result of the influencing factor sample B, well 12 is the result of dissociation solution B, well 13 is the dissociation result of the influencing factor sample C but no further 10% pyrolysis was used after dissociation, and well 14 is the result of dissociation solution C but no further 10% pyrolysis was used after dissociation. Detailed Implementation

[0051] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0052] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0053] The self-made standard antigens for H1N1 influenza, H3N2 influenza, and BV influenza used in this invention were all prepared by Jianda Biopharmaceutical (Nanjing) Co., Ltd., with batch numbers RS-AOD-241104, RS-ATD-241104, and RS-BVD-241104, respectively. The trivalent recombinant influenza protein vaccine (Sf9 cells) used in this invention was prepared by Jianda Biopharmaceutical (Nanjing) Co., Ltd., with batch number 202506001.

[0054] Example 1: A dissociation solution for adsorbing influenza vaccines with aluminum adjuvant

[0055] A dissociation solution for adsorbing influenza vaccines with aluminum adjuvants is composed of the following raw materials: potassium phosphate solution, diethanolamine, Triton X-100, Tween-20, and dodecyl dimethyl betaine (BS-12). The amounts of each raw material are shown in Table 1.

[0056] Table 1 Composition of the dissociation solution

[0057]

[0058] The preparation method is as follows:

[0059] 1. Preparation of mother liquors for each solution:

[0060] (1) 1M potassium phosphate solution: Weigh 18.26g of dipotassium hydrogen phosphate trihydrate and 2.72g of potassium dihydrogen phosphate, and dissolve them in 100ml of water.

[0061] (2) 20% v / v diethanolamine: Dissolve 2 ml of diethanolamine in 8 ml of water.

[0062] (3) 10% v / v TritonX-100: Dissolve 5 ml of TritonX-100 in 45 ml of water.

[0063] (4) 10% v / v Tween-20: Dissolve 1 ml of Tween-20 in 10 ml of water.

[0064] (5) 30% w / w dodecyl dimethyl betaine: purchased from Maclean, catalog number D848892.

[0065] 2. Preparation of dissociation solution:

[0066] Add water, 1M potassium phosphate solution, 20% v / v diethanolamine, 10% v / v Triton X-100, 10% v / v Tween-20, and 30% w / w dodecyl dimethyl betaine to centrifuge tubes according to the concentrations in Table 1, mix thoroughly to obtain the dissociation solution.

[0067] Example 2: A desorption method for influenza vaccine adsorbed by aluminum adjuvant

[0068] A desorption method for adsorbing influenza vaccines using aluminum adjuvants: Take 300 μL of influenza vaccine, centrifuge at 10000g for 5 minutes, discard 270 μL of supernatant, add 270 μL of dissociation solution, mix well, place at 37℃ for 3 hours, centrifuge at 10000g for 5 minutes, and take the supernatant.

[0069] Example 3: A one-way immune diffusion (SRID) assay

[0070] The SRID experiment in this invention includes the above-mentioned desorption method. Specific steps:

[0071] 1. Sample preparation:

[0072] (1) Preparation of test solution: Take 300 μL of influenza vaccine, centrifuge at 10000g for 5 minutes and discard 270 μL of supernatant. Add 270 μL of dissociation buffer and mix well. Place at 25-37℃ for 3-4 hours, centrifuge at 10000g for 5 minutes and take the supernatant. Mix the supernatant with 10% lysis reagent at a volume ratio of 9:1 and place at room temperature for 30 minutes for lysis. The lysed solution is used as the test solution.

[0073] (2) Preparation of standard solutions: Standard solutions of different concentrations were mixed with 10% lysis agent at a volume ratio of 9:1 and then placed at room temperature for 30 minutes for lysis. After lysis, the solutions were serially diluted and a standard curve was plotted.

[0074] The 10% lysis agent is prepared by dissolving 1g of Zwittergent 3-14 in 10ml of purified water as a 10% lysis agent.

[0075] 2. Preparation of agarose gel containing specific antiserum:

[0076] Take a suitable-sized culture dish, ensuring it is clean, and place it on a horizontal work surface. Add the appropriate amount of antiserum to 56°C 1.5% agarose, and gently shake to mix, avoiding the formation of air bubbles. The volume ratio of rabbit anti-BV serum to 1.5% agarose is 15 μL:1 mL, rabbit anti-H3N2 serum is 5 μL:1 mL, and rabbit anti-H1N1 serum is 10 μL:1 mL. Pour the mixed agarose into the culture dish, immediately removing any air bubbles that form. After cooling and solidifying at room temperature for 30 minutes, punch holes using a 3 mm punch, spacing each hole at least 15 mm apart. If any residual agarose remains in the holes, remove it completely using a pipette tip. The agarose should only be used on the same day.

[0077] 3. Sample loading and diffusion:

[0078] Add 10 μL of the above sample to each well of an agar gel containing specific antiserum, changing the pipette tip each time. After adding the sample, let it stand at room temperature for 1 hour, then transfer it to a humidified chamber and place it for 21 hours, for a total diffusion time of 22 hours.

[0079] 4. Glue washing and pressing:

[0080] After removing the gel from the culture dish, wash it with PBS for 15 minutes each time. Then, place it between two sheets of paper to absorb excess serum and water and press it with a heavy object for 15 minutes. Repeat this process four times until the agar gel becomes a thin layer.

[0081] 5. Dyeing and decolorization:

[0082] After rinsing the thinned agar gel twice with deionized water, it is then transferred to the staining solution and stained for about 30 minutes. After that, it is taken out, the surface staining solution is washed off with purified water, and then it is soaked in purified water to decolorize until the precipitate ring is clear.

[0083] 6. Data Processing:

[0084] After photographing the samples using an imager, the diameter of the precipitation rings was measured using ImageJ software. The diameter of each ring was measured three times in different directions, and the average value was taken. A linear regression was then performed with the precipitation ring diameter as the ordinate and the concentration of the standard as the abscissa.

[0085] Example 4 Methodological Validation

[0086] This invention also provides methodological validation of the above methods, including specificity, linearity and range, accuracy, precision and robustness, as detailed below:

[0087] 1. Specificity verification

[0088] 1.1 Sample Processing and Operation

[0089] Take 300 μL of each of the following self-made standard antigens: H1N1 influenza, H3N2 influenza, and BV influenza. Centrifuge at 10000g for 5 minutes, discard 270 μL of the supernatant, add 270 μL of dissociation buffer, mix well, and incubate at 25-37℃ for 3-4 hours. Centrifuge at 10000g for 5 minutes and collect the supernatant. Mix the supernatant with 10% lysis buffer at a volume ratio of 9:1 and incubate at room temperature for 30 minutes to perform lysis. Use the lysed solution as the test solution to obtain 30 μg / mL of each of the self-made standard antigens: H1N1 influenza, H3N2 influenza, and BV influenza.

[0090] 1.2 Preparation of agarose gel containing specific antiserum:

[0091] Agarose gels containing rabbit anti-H1N1 serum, rabbit anti-H3N2 serum, and rabbit anti-BV serum were obtained by referring to the method described in Example 3.

[0092] 1.3 Sample loading and diffusion:

[0093] Add 10 μL of the above-mentioned 30 μg / mL H1N1 influenza self-made standard antigen to the wells of an agar gel containing rabbit anti-H1N1 serum, changing the pipette tip each time. After adding the sample, let it stand at room temperature for 1 hour, then transfer it to a humidified chamber and place it for 21 hours, with a total diffusion time of 22 hours; at the same time, use 30 μg / mL H3N2 influenza self-made standard antigen and BV influenza self-made standard antigen as controls.

[0094] Add 10 μL of the above-mentioned 30 μg / mL H3N2 influenza self-made standard antigen to the wells of an agar gel containing rabbit anti-H1N1 serum, changing the pipette tip each time. After adding the sample, let it stand at room temperature for 1 hour, then transfer it to a humidified chamber and place it for 21 hours, with a total diffusion time of 22 hours; at the same time, use 30 μg / mL H1N1 influenza self-made standard antigen and BV influenza self-made standard antigen as controls.

[0095] Add 10 μL of the 30 μg / mL BV influenza self-made standard antigen to the wells of an agar gel containing rabbit anti-H1N1 serum, changing the pipette tip each time. After adding the sample, let it stand at room temperature for 1 hour, then transfer it to a humidified chamber and place it for 21 hours, for a total diffusion time of 22 hours; at the same time, use 30 μg / mL H1N1 influenza self-made standard antigen and H3N2 influenza self-made standard antigen as controls.

[0096] 1.4 Glue washing and pressing, dyeing and decolorization, data processing

[0097] The adhesive washing and pressing, dyeing and decolorization, and data processing were carried out in accordance with the method described in Example 3.

[0098] 1.5 Acceptable Standards

[0099] (1) Other types of influenza recombinant protein standards should be free of precipitation rings.

[0100] (2) The recovery rate of spiked samples should be between 70% and 130%, and the dissociation solution should not interfere with the detection results.

[0101] 1.6 Experimental Results

[0102] The experimental results are shown in Table 2 and Figure 1 As shown, specificity meets acceptable criteria.

[0103] Table 2 Results of specific spiking test

[0104]

[0105] 2. Linearity and Range

[0106] A series of self-made standard antigen solutions of H1N1 influenza (10-40 μg / mL), H3N2 influenza (10-40 μg / mL), and BV influenza (10-40 μg / mL) were prepared according to the method described in Example 3, and then tested. Linear regression was performed using the concentration of the standards and the corresponding precipitate ring diameter.

[0107] Acceptable criteria: Good linearity within this concentration range, with a linear correlation coefficient R0 on the standard curve. 2 ≥0.95.

[0108] The test results are shown in Tables 3-5. The linearity and range test results meet the acceptable standards.

[0109] Table 3. Results of linearity and range tests of the self-made standard antigen for H1N1 influenza.

[0110]

[0111] Table 4. Results of linearity and range tests of the self-made standard antigen for H3N2 influenza.

[0112]

[0113] Table 5. Results of linearity and range tests of the self-made standard antigen for influenza BV.

[0114]

[0115] 3. Accuracy

[0116] Prepare 30 μg / mL, 25 μg / mL, and 20 μg / mL self-made standard antigen solutions for H1N1 influenza, H3N2 influenza, and BV influenza, respectively, using the method described in Example 3. Prepare three samples of each concentration and perform detection as described in Example 3.

[0117] Acceptable standard: The recovery rate should be in the range of 70.0%-130.0%.

[0118] The measurement results are shown in Tables 6-8:

[0119] Table 6. Results of the accuracy test for the self-made standard antigen for H1N1 influenza.

[0120]

[0121] Table 7. Results of the accuracy test for the self-made standard antigen for H3N2 influenza.

[0122]

[0123] Table 8. Results of the accuracy test for the self-made standard antigen for influenza BV.

[0124]

[0125] 4. Repeatability

[0126] Trivalent recombinant influenza vaccine H1N1 type (Sf9 cells), trivalent recombinant influenza vaccine H3N2 type (Sf9 cells), and trivalent recombinant influenza vaccine BV type (Sf9 cells) were prepared according to the method described in Example 3. Six samples of each type were prepared and tested according to the method described in Example 3.

[0127] Acceptable standard: The RSD value of hemagglutinin content of each type in all six samples should be ≤20%.

[0128] The measurement results are shown in Table 9:

[0129] Table 9 Results of Repeatability Tests

[0130]

[0131] 5. Intermediate precision

[0132] On different days (Day A or Day B), two lab technicians (A or B) took the same batch of trivalent recombinant influenza protein vaccine H1N1 (Sf9 cells), trivalent recombinant influenza protein vaccine H3N2 (Sf9 cells), and trivalent recombinant influenza protein vaccine BV (Sf9 cells). They prepared 30 μg / mL H1N1 vaccine test samples, H3N2 vaccine test samples, and BV vaccine test samples, respectively, with six samples of each type. The samples were then tested according to the method described in Example 3.

[0133] Acceptable standard: The RSD value of hemagglutinin content of each type in 12 samples should be ≤20%.

[0134] The measurement results are shown in Table 10:

[0135] Table 10 Results of intermediate precision test

[0136]

[0137] 6. Durability

[0138] Trivalent recombinant influenza vaccine H1N1 (Sf9 cells), trivalent recombinant influenza vaccine H3N2 (Sf9 cells), and trivalent recombinant influenza vaccine BV (Sf9 cells) were prepared according to the method described in Example 3. Two samples of each type of vaccine (30 μg / mL) were prepared and tested according to the method described in Example 3. The effect of the test solution being placed at 2-8℃ for 24 h, 48 h, and 72 h on the test results was investigated.

[0139] Acceptable standard: The RSD value of samples placed for different times should be ≤20%.

[0140] The measurement results are shown in Table 11:

[0141] Table 11 Durability Test Results

[0142]

[0143] Experimental Example 1: Comparison with dissociation solutions in existing technologies

[0144] Comparative Example 1

[0145] Dissociation solution A in non-patent literature (Gao Qiang, Li Changgui, Liu Shuzhen, et al. Establishment of a method for detecting hemagglutinin content in aluminum-adsorbed influenza vaccines [J]. Chinese Journal of Biological Products, 2008, (01): 60-61+69.).

[0146] Dissociation solution A: 20% w / v trisodium citrate.

[0147] Dissociation method: After mixing equal volumes of dissociation solution A and the sample, dissociate at room temperature for 2 hours, centrifuge at 10000g for 5 minutes, and collect the supernatant. Use a one-way immunodiffusion assay to determine the content of each type of hemagglutinin in the supernatant.

[0148] Comparative Example 2

[0149] Dissociation solution B in non-patent literature (Gao Qiang, Li Changgui, Liu Shuzhen, et al. Establishment of a method for detecting hemagglutinin content in aluminum-adsorbed influenza vaccines [J]. Chinese Journal of Biological Products, 2008, (01): 60-61+69.).

[0150] Dissociation solution B: 8.55 mL PBS, 1.25 mL 20% diethanolamine, 0.2 mL 10% Triton X-100.

[0151] Dissociation method: After mixing equal volumes of dissociation solution B and the sample, dissociate at room temperature for 2 hours, centrifuge at 10000g for 5 minutes, and collect the supernatant. Use a one-way immunodiffusion assay to determine the content of each type of hemagglutinin in the supernatant.

[0152] Comparative Example 3

[0153] The optimal dissociation solution in patent document CN101971030 A is named dissociation solution C.

[0154] Dissociation solution C: 50 μL of 10% w / v Zwittergent 3-14 solution mixed with 950 μL of 350 mM K2HPO4 solution.

[0155] Dissociation method: Take 300 μL of influenza vaccine, centrifuge at 10000g for 5 minutes, discard 270 μL of supernatant, add 270 μL of dissociation buffer C, mix well, incubate at 2-8℃ for 18 hours, centrifuge at 10000g for 5 minutes, and collect the supernatant. Use a one-way immunodiffusion assay to determine the content of hemagglutinin of each type in the supernatant.

[0156] The dissociation solution and dissociation method of this invention:

[0157] The dissociation solution of this invention is composed of the following raw materials: potassium phosphate solution, diethanolamine, Triton X-100, Tween-20, and dodecyl dimethyl betaine (BS-12). The amounts of each raw material are shown in Table 1.

[0158] Desorption method: Take 300 μL of influenza vaccine, centrifuge at 10000g for 5 minutes, discard 270 μL of supernatant, add 270 μL of dissociation solution, mix well, place at 37℃ for 3 hours, centrifuge at 10000g for 5 minutes, and take the supernatant.

[0159] Comparison of dissociation effects:

[0160] The dissociation effect was compared with that described in Example 3 of this invention using a single-path immune diffusion (SRID) test. The theoretical value of each type of hemagglutinin in the sample was 30 μg / ml. The test results are as follows: Figure 2 As shown in Tables 12 and 13.

[0161] Figure 2 Wells 1-5 represent the standard curve (well 1: 40 μg / mL, well 2: 30 μg / mL, well 3: 25 μg / mL, well 4: 20 μg / mL, well 5: 10 μg / mL). Well 6 is a positive control. Well 7 shows the dissociation result of the influencing factor sample (C). Well 8 shows the result of the dissociation solution (C). Well 9 shows the dissociation result of the influencing factor sample (A). Well 10 shows the result of the dissociation solution (A). Well 11 shows the dissociation result of the influencing factor sample (B). Well 12 shows the result of the dissociation solution (B). Samples in wells 13 and 14 are the same as those in wells 7 and 8, but after dissociation, samples in wells 13 and 14 are directly loaded without further 10% lysis using the lysis reagent. The results from both methods are similar. The diameter of the influencing factor sample is smaller than the lowest point of the standard curve (10 μg / mL).

[0162] Dissociation solutions A and B were not effective at dissociating the three types of hemagglutinin. As can be seen from the figure, the results of dissociation solutions A (well 10) and B (well 12) were both smaller than the diameter of the lowest point of the standard curve (10 μg / mL), so the results could not be calculated.

[0163] The results of dissociation solution C and the dissociation solution in this invention are shown in Tables 12-13 below:

[0164] Table 12 Measurement Results

[0165]

[0166] Table 13 Recovery rate of the determination results

[0167]

[0168] The dissociation solution provided by this invention has good dissociation effect, and the detection results of the content of hemagglutinin of various types are close to the theoretical value. The recovery rate is between 90% and 110%, while the recovery rate of dissociation solution C is less than 50%.

[0169] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A dissociation solution for adsorbing influenza vaccines with aluminum adjuvant, characterized in that, The dissociation solution consists of 0.4 mol / L-0.5 mol / L potassium phosphate solution, 2%-3% v / v diethanolamine, 0.4% v / v Triton X-100, 0.2% v / v Tween-20, 0.4% w / w dodecyl dimethyl betaine, and water.

2. The dissociation solution according to claim 1, characterized in that, The dissociation solution consists of 0.5 mol / L potassium phosphate solution, 2.5% v / v diethanolamine, 0.4% v / v Triton X-100, 0.2% v / v Tween-20, 0.4% w / w dodecyl dimethyl betaine and water.

3. A method for antigen dissociation in influenza vaccines adsorbed with aluminum adjuvants, characterized in that, The antigen dissociation method includes using the dissociation solution according to any one of claims 1-2.

4. The antigen dissociation method according to claim 3, characterized in that, The antigen dissociation method includes the following steps: adsorbing influenza vaccine with aluminum adjuvant, centrifuging, discarding the supernatant, adding dissociation solution to the precipitate, incubating, centrifuging, taking the supernatant, and obtaining the dissociated antigen.

5. The antigen dissociation method according to claim 4, characterized in that, The volume ratio of the precipitate to the dissociation solution is 1:8-10.

6. The antigen dissociation method according to claim 5, characterized in that, The volume ratio of the precipitate to the dissociation solution is 1:

9.

7. The antigen dissociation method according to claim 4, characterized in that, The incubation process involves placing the food at 25-37°C for 3-4 hours.

8. The antigen dissociation method according to claim 7, characterized in that, The incubation process involves placing the food at 37°C for 3 hours.

9. A method for detecting the antigen content in an influenza vaccine adsorbed with aluminum adjuvant, characterized in that, The detection method includes a pretreatment method for adsorbing influenza vaccines with aluminum adjuvants, wherein the pretreatment method includes using the dissociation solution according to any one of claims 1-2 to dissociate the antigens in the influenza vaccines adsorbed by the aluminum adjuvants.

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