CN111812313B - Dissociation method of antigen in aluminum adjuvant adsorption type novel coronavirus inactivated vaccine - Google Patents
Dissociation method of antigen in aluminum adjuvant adsorption type novel coronavirus inactivated vaccine Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The invention provides a dissociation method of an antigen in an aluminum adjuvant adsorption type novel coronavirus inactivated vaccine. The desorption agent composition adopted by the method comprises sucrose, magnesium chloride, potassium phosphate and polysorbate-80; the dosage ratio of the sucrose, the magnesium chloride, the potassium phosphate and the polysorbate-80 is (50-100) g: (2-6) g: (0.3-0.6) mol: (0.5-1) ml. The method is used for dissociating the antigen in the novel aluminum adjuvant adsorption type coronavirus inactivated vaccine, can stabilize a solution system, protect the immune activity of the antigen, destroy the adsorption force of the aluminum adjuvant, and quickly separate the antigen from the aluminum adjuvant; eliminating the interference of the aluminum adjuvant on the accurate quantification of the antigen; the method can solve the problem that the biological structure of the epitope of the new coronavirus is unstable in the dissociation process, has the characteristics of good repeatability, high accuracy and strong specificity, and provides an important tool for evaluating the key quality attribute of the novel coronavirus inactivated vaccine.
Description
Technical Field
The invention belongs to the field of biological pharmacology, and particularly relates to a dissociation method of an antigen in an aluminum adjuvant adsorption type novel coronavirus (SARS-CoV-2) inactivated vaccine.
Background
The challenge of global new crown epidemic situation is still serious, and China is tightening scientific research and development of new crown vaccines and marketing of products. A novel coronavirus (SARS-CoV-2) inactivated vaccine is a powerful shield for resisting virus invasion, the key component of the inactivated vaccine is an antigen associated with clinical effectiveness, and the quality attribute effect of a product can be evaluated only by accurate quantification in the quality control process of the product.
The existing research considers that the new corona vaccine antigen mainly comprises S protein, M protein, N protein and the like, and can only be detected by an enzyme-linked immunosorbent assay. Currently, the technical route of inactivated SARS-CoV-2 vaccine reported to enter clinical research stage globally uses aluminum adjuvant to enhance immunity (Qiang G, Linlin B, Haiyan Met al. Rapid degradation of an inactivated vaccine for SARS-CoV-2.science. DOI:10.1126/science. abc1932; Hui W, Yuntao Z, Baoying H et al. development of an inactivated vaccine candidate, BBIBP-CorV, with potential protection against SARS-CoV-2.cell. https:// doi. org/10.1016/j. cell. 2020.06.008). Although the immunogenicity and antigen conservation of the vaccine can be remarkably improved by using the aluminum adjuvant, the aluminum adjuvant has high surface area structure characteristic, so that the vaccine has high adsorption capacity, and the formed adjuvant-antigen adsorbate causes that specific binding sites of most antigens are deeply buried in aluminum adjuvant particle precipitates, so that the content of the antigens cannot be directly detected by using an enzyme-linked immunosorbent assay. Meanwhile, the existing research considers that the potential key antigen of the new coronavirus is positioned on the virus envelope, is easily influenced by various factors such as solution property, storage condition, fluid power and the like, has unstable structure, is easy to denature in the violent dissociation process, and cannot detect the antigenicity once the biological structure of the antigen is destroyed. And the extreme solution system possibly generated in the process of antigen dissociation can also influence the accuracy of detection. The main mechanism of the aluminum adjuvant for adsorbing the antigen is acting force such as electrostatic attraction; although aluminum hydroxide adjuvant can be dissolved in acid or alkaline conditions and neutral solutions of alpha-hydroxy acids such as citric acid and malic acid, the antigen protein structure is not protected to a certain extent in advance, and the method is not suitable for dissociation of new coronavirus antigen with unstable structure. These difficulties are the key technical bottleneck in antigen quality control during the development of SARS-CoV-2 inactivated vaccine.
Therefore, it is necessary to develop a dissociation method of antigen and aluminum adjuvant specificity aiming at quantitative detection of effective components of the new corona vaccine, so that the dissociation method can stabilize a solution system, protect the immunological activity of the antigen, destroy the adsorption force of the aluminum adjuvant, and quickly separate the antigen from the aluminum adjuvant, and simultaneously, the structural specificity of the virus antigen epitope is not influenced, thereby realizing accurate quantitative detection of the antigen in the vaccine. At present, no related report of the dissociation method of the effective components of the aluminum adjuvant adsorption type SARS-CoV-2 inactivated vaccine exists at home and abroad.
Disclosure of Invention
The invention aims to provide a desorbent composition for antigen dissociation in an aluminum adjuvant adsorption type novel coronavirus inactivated vaccine, which can dissociate antigens in the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine, and can stabilize a solution system, protect the immunological activity of the antigens, destroy the adsorption force of the aluminum adjuvant and quickly separate the antigens from the aluminum adjuvant in the process; the second purpose of the invention is to provide a kit for antigen dissociation in the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine; the third purpose of the invention is to provide a dissociation method of antigen in the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine, which is applied together with the desorbent composition, aims to rapidly and efficiently dissociate the antigen from the surface of the aluminum adjuvant crystal, keeps the complete biological structure of the aluminum adjuvant crystal, and can be used for effective quantitative detection, thereby achieving the aim of controlling the quality attribute of the product; the fourth purpose of the invention is to obtain the novel coronavirus antigen desorption solution by using the dissociation method, wherein the novel coronavirus antigen desorption solution has uniform, stable and mild solution characteristics, does not need special treatment, and can be directly suitable for the quantitative detection of the antigen by an enzyme-linked immunosorbent assay.
The purpose of the invention is realized by the following technical means:
in one aspect, the invention provides a desorbent composition for antigen dissociation in an aluminum adjuvant adsorption type novel coronavirus inactivated vaccine, wherein the desorbent composition comprises sucrose, magnesium chloride, potassium phosphate salt and polysorbate-80;
the dosage ratio of the sucrose to the magnesium chloride to the potassium phosphate to the polysorbate-80 is (50-100) g: (2-6) g: (0.3-0.6) mol: (0.5-1) ml.
In a preferred embodiment of the present invention, magnesium chloride hexahydrate may be used as the magnesium chloride.
In the desorbent composition described above, preferably, the potassium phosphate salt comprises a mixture of potassium phosphate salts consisting of dipotassium hydrogen phosphate (anhydrous) and potassium dihydrogen phosphate.
In the desorbent composition described above, preferably the molar ratio of the dipotassium hydrogen phosphate (anhydrous) to the potassium dihydrogen phosphate is 4: 1.
On the other hand, the invention also provides a kit for antigen dissociation in the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine, wherein the kit comprises sucrose, magnesium chloride, potassium phosphate and polysorbate-80;
when the kit is used for dissociating the antigen in the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine, the final mass concentration of the sucrose in a dissociation system is 5-10% (w/v); the mass final concentration of the magnesium chloride is 0.2-0.6% (w/v); the final molar concentration of the potassium phosphate is 0.3-0.6 mol/L; the volume final concentration of the polysorbate-80 is 0.05-0.1% (v/v).
The final concentration of the sucrose, the magnesium chloride, the potassium phosphate salt and the polysorbate-80 in the invention is the mass concentration, the molar concentration or the volume concentration of the 4 raw materials in a dissociation system formed by adding the raw materials into the novel coronavirus inactivated vaccine adsorbed by the aluminum adjuvant.
In still another aspect, the present invention provides a method for dissociating an antigen in an aluminum adjuvant adsorption type novel inactivated coronavirus vaccine, comprising the following steps:
adding the desorbent composition or adopting the kit to dissociate the antigens of the novel coronavirus aiming at the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine to obtain the novel coronavirus antigen desorption solution.
In the above method, preferably, the novel coronavirus is SARS-CoV-2.
In the above method, preferably, the desorbent composition is added to the aluminum-adjuvant-adsorbed novel inactivated coronavirus vaccine, and the dissociation of the antigen of the novel coronavirus comprises:
adding sucrose into the novel aluminum adjuvant adsorption type coronavirus inactivated vaccine;
after the sucrose is dissolved, adding magnesium chloride (preferably magnesium chloride hexahydrate or a magnesium chloride hexahydrate solution);
after mixing uniformly, sequentially adding potassium phosphate (preferably, a potassium phosphate buffer solution can be adopted) and polysorbate-80, and dissociating the target antigen to obtain a novel coronavirus antigen desorption solution;
the dissociation process is constant temperature incubation, and at least one ultrasonic treatment is carried out during the incubation period.
In the method, the magnesium chloride hexahydrate solution is prepared by adding water into magnesium chloride hexahydrate, and the mass concentration of the magnesium chloride hexahydrate solution is 1-2%; the potassium phosphate buffer solution is prepared by adding water into potassium phosphate, and the molar concentration of the potassium phosphate buffer solution is 0.6-1.2 mol/L; wherein, the water used in the preparation process comprises purified water and/or water for injection.
The potassium phosphate buffer of the present invention is prepared by the following method (the solvent is exemplified by water for injection):
(1)1mol/L potassium phosphate salt buffer: 13.95g of dipotassium phosphate (anhydrous) and 2.69g of monopotassium phosphate are weighed, and after the dipotassium phosphate and the monopotassium phosphate are completely dissolved by using water for injection, the volume is determined to be 100ml of a volumetric flask, and the obtained solution is the potassium phosphate buffer solution with the concentration of 1 mol/L.
(2)0.6mol/L or less molar concentration <1mol/L potassium phosphate buffer: is prepared by mixing 1mol/L potassium phosphate buffer solution and water for injection according to a proportion. Taking 10ml and 0.6mol/L potassium phosphate buffer solution as an example, 6ml and 1mol/L potassium phosphate buffer solution and 4ml water for injection are measured and mixed uniformly to obtain the potassium phosphate buffer solution with the concentration of 0.6 mol/L.
(3)1mol/L < molar concentration ≤ 1.2mol/L potassium phosphate buffer: is prepared by mixing 2mol/L potassium phosphate buffer solution and water for injection according to a proportion. 27.9g of dipotassium phosphate (anhydrous) and 5.39g of monopotassium phosphate are weighed, and after the dipotassium phosphate and the monopotassium phosphate are completely dissolved by using water for injection, the volume is determined to be 100ml of a volumetric flask, and the obtained solution is 2mol/L potassium phosphate buffer solution. Taking 10ml of 1.2mol/L potassium phosphate buffer solution as an example, 6ml of 2mol/L potassium phosphate buffer solution and 4ml of water for injection are measured and mixed uniformly to obtain 1.2mol/L potassium phosphate buffer solution.
In the dissociation process, the effect of stabilizing the stability of the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine can be achieved by adding the sucrose and the magnesium chloride, and the virus antigen can be effectively protected. In the above method, preferably, the final mass concentration of the added sucrose is 5-10%; the final mass concentration of the added magnesium chloride is 0.2-0.6%; the final molar concentration of the potassium phosphate salt is 0.3-0.6 mol/L; the volume final concentration of the polysorbate-80 is 0.05-0.1%.
In the method, preferably, in the dissociation process, the incubation temperature at constant temperature (which can be water bath or air bath) is 4-25 ℃, and the incubation time at constant temperature is 20-240 min; for example: 20min, 60min, 100min, 200min, 240min and the like.
In the method, preferably, the time of ultrasonic treatment in the constant-temperature incubation is 0.1-0.5 min, the ultrasonic frequency is 30-40 kHz, and the number of ultrasonic treatment is 1-3; for example: 1, 2 and 3 times.
In the above method, preferably, the method for preparing the aluminum adjuvant adsorption type novel inactivated coronavirus vaccine comprises:
diluting the virus stock solution of the novel coronavirus inactivated vaccine, and then adding an aluminum adjuvant to prepare the novel coronavirus antigen adsorption type novel coronavirus inactivated vaccine with the final concentration of 4-32U/ml and the final concentration of 0.2-0.85 mg/ml.
In the method, preferably, the antigen content of the virus stock solution of the novel inactivated coronavirus vaccine is more than or equal to 40U/ml, the total protein content is more than or equal to 100 mu g/ml, and the pH value is 5-8. The novel coronavirus inactivated vaccine stock solution also comprises a Vero cell DNA residual quantity, a Vero cell protein residual quantity, a bovine serum albumin residual quantity, a nuclease residual quantity and a beta-propiolactone residual quantity, which all meet the quality standard requirements of the inactivated vaccine stock solution.
The virus stock solution of the raw material, namely the novel coronavirus inactivated vaccine, can be prepared by using a novel coronavirus (SARS-CoV-2) by adopting a conventional virus culture method and an inactivation method.
In the above method, preferably, the aluminum adjuvant comprises aluminum hydroxide.
In still another aspect, the present invention also provides a novel coronavirus antigen desorption solution obtained by antigen dissociation according to the above method.
The invention has the beneficial effects that:
the invention provides a special desorbent composition with strong specificity for dissociating an effective component antigen in an aluminum adjuvant adsorption type novel coronavirus inactivated vaccine and a dissociation method, and in the process, a solution system can be stabilized, the antigen immunocompetence can be protected, the aluminum adjuvant adsorption force can be destroyed, and the antigen can be rapidly separated from an aluminum adjuvant; the dissociation method can eliminate the interference of the aluminum adjuvant on the detection of the antigen content in the sample, can solve the problem of unstable biological structure of the new coronavirus antigen in the dissociation process, has the characteristics of good repeatability, high accuracy and strong specificity, and provides an important tool for the evaluation of the key quality attribute of the novel coronavirus inactivated vaccine.
Drawings
FIG. 1 is a layout of microplate for reference substances and samples to be tested in example 1 of the present invention.
FIG. 2 is a graph showing the results of in vitro relative efficacy calculation by the double parallel line method in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1:
the embodiment provides a dissociation method of an antigen in an aluminum adjuvant adsorption type novel coronavirus inactivated vaccine, which specifically comprises the following steps:
(1) preparing a sample to be tested and a reference substance of the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine:
the sample to be tested of the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine is self-made (no commercial product is available at present). The preparation scheme is as follows: diluting a virus stock solution (the antigen content is 138U/ml and the protein content is 215 mug/ml) of the novel coronavirus inactivated vaccine according to a target concentration for preparing 8U/ml antigen in proportion, and then adding an aluminum adjuvant to prepare an aluminum adjuvant adsorption type SARS-CoV-2 inactivated vaccine sample with the final concentration of the novel coronavirus antigen of 8U/ml and the final concentration of the aluminum hydroxide adjuvant of 0.45 mg/ml.
The reference product is made by the manufacturer (no commercial product is available at present). The preparation scheme is as follows: the virus stock solution (in the detection index of the virus stock solution, the antigen content of the stock solution detection index is 171U/ml, and the protein content is 287 mu g/ml) of the novel coronavirus inactivated vaccine is diluted according to the target concentration for preparing 8U/ml antigen in proportion, then an aluminum adjuvant is added to prepare a SARS-CoV-2 inactivated vaccine reference product with the final concentration of the novel coronavirus antigen of 8U/ml and the final concentration of the aluminum hydroxide adjuvant of 0.45mg/ml, the SARS-CoV-2 inactivated vaccine reference product is produced according to national standards, and the product with qualified titer is calibrated in an animal body through physicochemical and biological tests.
(2) The composition of the desorbent composition in this example includes: sucrose, magnesium chloride hexahydrate, potassium phosphate salt (mixed potassium phosphate salt consisting of anhydrous dipotassium hydrogen phosphate and potassium dihydrogen phosphate) and polysorbate-80, all of which are commercially available.
In the following desorption process of this example, a magnesium chloride hexahydrate solution and a potassium phosphate buffer solution were used;
the specific preparation process of the magnesium chloride hexahydrate solution with the mass concentration of 2 percent is as follows:
weighing 2g of magnesium chloride hexahydrate, completely dissolving the magnesium chloride hexahydrate by using injection water, and then fixing the volume to a 100ml volumetric flask to obtain a solution, namely a magnesium chloride hexahydrate solution with the mass concentration of 2%.
The specific preparation process of the 1mol/L potassium phosphate buffer solution comprises the following steps:
13.95g of dipotassium phosphate (anhydrous) and 2.69g of monopotassium phosphate are weighed, and after the dipotassium phosphate and the monopotassium phosphate are completely dissolved by using water for injection, the volume is determined to be 100ml of a volumetric flask, and the obtained solution is the potassium phosphate buffer solution with the concentration of 1 mol/L.
(3) The dissociation procedure was carried out:
adding sucrose into the novel aluminum adjuvant adsorption type coronavirus inactivated vaccine; after the sucrose is dissolved, adding a magnesium chloride hexahydrate solution with the mass concentration of 2%; after mixing evenly, sequentially adding 1mol/L potassium phosphate buffer solution and polysorbate-80; in the dissociation system, the sucrose mass final concentration is 10% (w/v), the magnesium chloride hexahydrate mass final concentration is 1% (w/v), the potassium phosphate salt molar final concentration is 0.5mol/L, and the polysorbate-80 volume final concentration is 0.05% (v/v).
After being mixed evenly, the mixture is placed at the constant temperature of 25 ℃ for incubation and dissociation, the duration is 60min, ultrasonic treatment is carried out for 1 time in the incubation period, the ultrasonic time is 0.2min, the ultrasonic frequency is 40kHZ, and the novel coronavirus antigen desorption solution is obtained after dissociation.
(4) Detecting the antigen content in the novel coronavirus antigen desorption solution:
the novel coronavirus antigen desorption solution was diluted at 1 ×, 2 ×, 4 ×, 8 ×. And detecting by adopting an enzyme-linked immunosorbent assay antigen kit, and loading a reference substance and a sample to be detected into a plurality of wells according to the concentration gradient (the layout of an ELISA plate is shown in figure 1). The method is characterized in that the basic principle of a biological assay double parallel line method is adopted to carry out in-vitro relative efficacy statistical analysis, the efficacy of the vaccine to be detected relative to a reference product is calculated, the detection result is 1.026 (shown in table 1, table 2 and figure 2), and the qualification standard is 0.5-2.0.
Table 1 is the results of in vitro relative potency data calculated by the double parallel line method.
Table 1:
table 2 is a statistical result of in vitro potency data calculated for the double parallel line method.
Table 2:
example 2:
the results of antigen dissociation in SARS-CoV-2 inactivated vaccine samples of different quality attributes are provided in this example, and the applicability of the dissociation method to different samples is examined. The method specifically comprises the following steps:
(1) preparing a sample to be tested and a reference substance:
the process for preparing the samples to be tested is the same as in example 1, the concentration preparation is shown in table 3, and the corresponding reference substances are prepared by the same method as in example 1.
(2) Preparation of desorbent composition: the same as in example 1.
(3) The dissociation procedure was carried out: the same as in example 1.
(4) The method for detecting the antigen content in the novel coronavirus antigen desorption solution is the same as that in example 1, and the specific dilution method is shown in table 3.
Table 3 shows the detection results of SARS-CoV-2 inactivated vaccine antigens with different quality attributes.
Table 3:
note: the reference substance adopts a synchronous dissociation method and a detection method.
As can be seen from the experimental results of table 3: the dissociation method of the invention has good applicability to the dissociation of the aluminum adjuvant adsorption type SARS-CoV-2 inactivated vaccine antigen.
Example 3:
the results of dissociation of the antigen in the inactivated SARS-CoV-2 vaccine by the dissociation method under different concentrations of the desorbent composition and different dissociation procedure parameter conditions are provided in the present example, and the applicability of the method to samples is examined. The method specifically comprises the following steps:
(1) preparing a sample to be tested and a reference substance: the same as in example 1.
(2) Preparation of desorbent composition:
the mass final concentration of the sucrose is 5-10% (w/v).
The final mass concentration of the magnesium chloride hexahydrate is 0.5-1% (w/v).
The preparation method of 0.6mol/L potassium phosphate buffer solution comprises the following steps:
is prepared by mixing 1mol/L potassium phosphate buffer solution and water for injection according to a proportion. Taking 10ml of 0.6mol/L potassium phosphate buffer solution as an example, 6ml of 1mol/L potassium phosphate buffer solution and 4ml of water for injection are measured and mixed uniformly to obtain 0.6mol/L potassium phosphate buffer solution.
1mol/L potassium phosphate buffer solution preparation: the same as in example 1.
The preparation method of 1.2mol/L potassium phosphate buffer solution comprises the following steps:
is prepared by mixing 2mol/L potassium phosphate buffer solution and water for injection according to a proportion. 27.9g of dipotassium phosphate (anhydrous) and 5.39g of monopotassium phosphate are weighed, and after the dipotassium phosphate and the monopotassium phosphate are completely dissolved by using water for injection, the volume is determined to be 100ml of a volumetric flask, and the obtained solution is 2mol/L potassium phosphate buffer solution. Taking 10ml and 1.2mol/L potassium phosphate buffer solution as an example, 6ml and 2mol/L potassium phosphate buffer solution and 4ml water for injection are measured and mixed uniformly to obtain the 1.2mol/L potassium phosphate buffer solution.
The final molar concentration of the potassium phosphate salt is 0.3 mol/L-0.6 mol/L.
The polysorbate-80 volume final concentration is 0.05-0.1% (v/v).
(3) The dissociation procedure was carried out as shown in table 4.
(4) Antigen determination: the same as in example 1.
Table 4 shows the results of the detection of SARS-CoV-2 inactivated vaccine by different dissociation methods.
Table 4:
note: the reference substance adopts a synchronous dissociation method and a detection method.
As can be seen from the experimental results of table 2: the method has good applicability to the dissociation of the antigen of the aluminum adjuvant adsorption type SARS-CoV-2 inactivated vaccine.
Example 4:
this example examines the specificity of the dissociation method, i.e., to assess whether the dissociation method interferes with the detection of the antigen. The method specifically comprises the following steps:
(1) preparing a sample to be tested: and preparing a diluent, and preparing the virus stock solution of the novel coronavirus inactivated vaccine and the aluminum adjuvant into a sample to be detected according to the final concentration of 0U/ml (without adding the virus stock solution of the novel coronavirus inactivated vaccine) and 0.45mg/ml respectively.
(2) Preparation of desorbent composition: the same as in example 3.
(3) The dissociation procedure was carried out: the same as in example 1.
(4) Antigen determination: the same as in example 1. The results are shown in Table 5.
Table 5 shows the results of investigation of specificity of the dissociation method of SARS-CoV-2 inactivated vaccine antigen.
Table 5:
as can be seen from the experimental results of table 5: the desorbent composition has no interference to antigen detection, and the method has good specificity. It is demonstrated that the dissociation method of the invention is applicable to the dissociation of SARS-CoV-2 inactivated vaccine antigen.
Example 5:
this example examines the reproducibility of the dissociation method and verifies the stability of the method for dissociating SARS-CoV-2 inactivated vaccine antigen. The method specifically comprises the following steps:
(1) preparing a sample to be tested and a reference substance: the same as in example 1.
(2) Preparation of desorbent composition: the same as in example 1.
(3) The dissociation procedure was carried out: the same as in example 1.
(4) Antigen determination: the same as in example 1. The results of dissociation of the antigen of the inactivated vaccine of SARS-CoV-2 are shown in Table 6.
Table 6 shows the results of repeated detection of the dissociation method of the SARS-CoV-2 inactivated vaccine antigen.
Table 6:
note: the reference substance adopts a synchronous dissociation method and a detection method.
As can be seen from the experimental results of table 6: the aluminum adjuvant adsorption type SARS-CoV-2 inactivated vaccine antigen dissociation method has good repeatability.
The invention adopts a special desorption agent composition and a procedure thereof to dissociate the aluminum adjuvant adsorption type SARS-CoV-2 inactivated vaccine, and the result shows that the dissociation method can dissociate the target effective components of the new crown inactivated vaccine and keep complete antigenicity, and the RSD value of the antigen repeatability determination after dissociation is 8.3%; the method is proved to have good repeatability.
Comparative example 1:
this comparative example illustrates comparative experiments in the literature reporting the dissociation of other desorbents for antigens of novel inactivated coronavirus vaccines. The method specifically comprises the following steps:
(1) preparing a sample to be tested and a reference substance: the same as in example 1.
(2) Desorbent and dissociation implementation process: see table 7.
(3) Antigen determination: the same as in example 1. The results of the antigen dissociation comparison of the inactivated vaccine against SARS-CoV-2 are shown in Table 7.
Table 7 shows the effect of different dissociation methods on the dissociation of the antigen of the inactivated SARS-CoV-2 vaccine.
Table 7:
as can be seen from Table 7, the dissociation method reported is not suitable for the antigen dissociation of the aluminum-adjuvant-adsorbed novel inactivated coronavirus vaccine.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (11)
1. A desorption agent composition for antigen dissociation in an inactivated vaccine aiming at an aluminum adjuvant adsorption type novel coronavirus SARS-CoV-2 comprises sucrose, magnesium chloride, potassium phosphate salt, polysorbate-80 and water;
the dosage ratio of the sucrose to the magnesium chloride to the potassium phosphate to the polysorbate-80 is (50-100) g: (2-6) g: (0.3-0.6) mol: (0.5-1) ml;
the potassium phosphate salt is a potassium phosphate salt mixture consisting of dipotassium hydrogen phosphate and potassium dihydrogen phosphate; the molar ratio of the dipotassium hydrogen phosphate to the potassium dihydrogen phosphate is 4: 1.
2. A kit for antigen dissociation in an inactivated vaccine against the aluminum-adjuvant-adsorbed novel coronavirus SARS-CoV-2, the kit comprising the desorbent composition of claim 1.
3. A dissociation method of antigen in inactivated vaccine of aluminum adjuvant adsorption type novel coronavirus SARS-CoV-2, which comprises the following steps:
adding the desorbent composition of claim 1 or the kit of claim 2 to the aluminum adjuvant-adsorbed novel coronavirus inactivated vaccine to dissociate the antigen of the novel coronavirus to obtain a novel coronavirus antigen desorption solution.
4. The method according to claim 3, wherein the desorption agent composition is added to the aluminum-adjuvant-adsorbed novel inactivated coronavirus vaccine, and the dissociation of the antigen of the novel coronavirus comprises:
adding sucrose into the novel aluminum adjuvant adsorption type coronavirus inactivated vaccine;
after the sucrose is dissolved, adding magnesium chloride;
after mixing uniformly, sequentially adding potassium phosphate and polysorbate-80, and dissociating the target antigen to obtain a novel coronavirus antigen desorption solution;
the dissociation process is constant temperature incubation, and at least one ultrasonic treatment is carried out during the incubation period.
5. The method of claim 4, wherein the sucrose is added at a final concentration of 5% to 10% by mass; the magnesium chloride is added with a mass final concentration of 0.2-0.6%; the final molar concentration of the potassium phosphate salt is 0.3-0.6 mol/L; the polysorbate-80 is added to the mixture in a final volume concentration of 0.05-0.1%.
6. The method according to claim 4, wherein the temperature of the constant temperature incubation is 4-25 ℃ and the time of the constant temperature incubation is 20-240 min during the dissociation process.
7. The method according to claim 6, wherein the time of the ultrasonic treatment in the constant-temperature incubation period is 0.1-0.5 min, the ultrasonic frequency is 30-40 kHz, and the number of ultrasonic treatments is 1-3.
8. The method according to claim 3, wherein the aluminum adjuvant adsorption type novel inactivated coronavirus vaccine is prepared by a method comprising the following steps of:
diluting the virus stock solution of the novel coronavirus inactivated vaccine, and then adding an aluminum adjuvant to prepare the novel coronavirus antigen adsorption type novel coronavirus inactivated vaccine with the final concentration of 4-32U/ml and the final concentration of 0.2-0.85 mg/ml.
9. The method according to claim 8, wherein the antigen content of the virus stock solution of the novel inactivated coronavirus vaccine is not less than 40U/ml, the total protein content is not less than 100 μ g/ml, and the pH value is 5-8.
10. The method of claim 8, wherein the aluminum adjuvant comprises aluminum hydroxide.
11. A novel antigen desorption solution for coronavirus SARS-CoV-2, which is obtained by the antigen dissociation method according to any one of claims 3 to 10.
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| CN112546213A (en) * | 2020-12-31 | 2021-03-26 | 中国医学科学院医学生物学研究所 | Method for preparing novel coronavirus vaccine and evaluation method aiming at effectiveness of novel coronavirus vaccine |
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