CN114594257A - Desorption composition of CpG ODN-containing adsorption type vaccine and application thereof - Google Patents
Desorption composition of CpG ODN-containing adsorption type vaccine and application thereof Download PDFInfo
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Abstract
The invention provides a desorption composition of an adsorption type vaccine containing CpG ODN, wherein the desorption composition comprises potassium phosphate, M199 culture medium, magnesium chloride and Triton-100. In another aspect, the present invention provides a kit for an adsorptive vaccine comprising a CpG ODN adjuvant, comprising the above-described desorbing composition. In another aspect, the present invention provides a method for in vitro testing the efficacy of an adsorbed vaccine comprising a CpG ODN adjuvant. In another aspect, the invention provides the composition, the kit and the detection method, and the application of the composition, the kit and the detection method in the desorption and in-vitro efficacy detection of the CpG ODN adjuvant-containing adsorption type vaccine. The desorption composition provided by the invention fills the blank in the aspect of antigen dissociation of the adsorption type vaccine containing the CpG ODN adjuvant, provides a rapid and accurate method for determining the efficacy of the adsorption type vaccine containing the CpG ODN adjuvant, and has pioneering significance.
Description
Technical Field
The invention relates to the determination of the efficacy of adjuvant-containing vaccines, in particular to a desorption composition in an adsorption-type vaccine containing CpG ODN and a method for determining the efficacy of an antigen in the adsorption-type vaccine containing CpG ODN.
Background
The adjuvant is an important component of various human vaccines, and the immune response of organisms to antigens can be enhanced by adding the adjuvant into the vaccines. The aluminum adjuvant is the earliest and most widely used vaccine adjuvant. The adsorption of the aluminum adjuvant to the antigen is influenced by various factors such as its own physicochemical properties, ionic strength of a solution, and concentration of the antigen, which enhances immune response through antigen reservoir effect, stimulation of dendritic cell maturation and T cell differentiation, promotion of release of dangerous signal molecules, and the like.
CpG ODN (CpG oligonucleotide) is representative of novel adjuvants and binds to Toll-like receptors on cell membranes to induce the production of various cytokines. CpG ODN, when bound to TLR9, eventually activates a variety of transcription factors including NF-. kappa. B, AP1, CEBP and CREB via MYD88, IRAK and TRAF 6. These transcription factors directly up-regulate cytokine and chemokine gene expression. CpG ODN starts immune stimulation through activating these cells, finally lead to the cascade reaction of natural killer cell (NK) indirect maturation, differentiation and proliferation, T cells and monocyte/macrophage secrete cytokine and chemotactic factor together, produce proinflammatory factor (IL-1, IL-6, IL-18 and TNF) and Th1 biased immunity (IFN gamma, IL-12) CpG ODN have the advantages of stability, low cost, easy synthesis, high efficiency and low toxicity, have the preference of inducing Th1 immune response, can prolong the persistence of producing antibody, and is an adjuvant widely applied in vaccine research.
The CpG ODN can be used together with other adjuvants to exert higher bioavailability, and the use conditions mainly comprise a CpG ODN single adjuvant, a CpG ODN adjuvant-hydrogenated alumina or an MF59 adjuvant and the like. The CpG ODN composite adjuvant composed of the nano aluminum adjuvant, the CpG ODN adjuvant and other related auxiliary materials improves the antigen uptake of antigen presenting cells, activates the functional maturation of the antigen presenting cells, and generates cytokines and chemokines so as to increase the immunogenicity of antigens.
The enzyme-linked immunosorbent assay is still one of the simplest and most effective antigen detection methods at present. However, due to the interaction force between the antigen and the CPG ODN adjuvant compound, the formed adjuvant-antigen adsorbate precipitate cannot be directly detected by using an enzyme-linked immunosorbent assay.
The main mechanism of the CPG ODN adjuvant compound for adsorbing the antigen is the force such as electrostatic attraction and hydrophobic effect, and partial substances such as metal ion chelating agent and surfactant can destroy the interaction force between the antigen and the adjuvant in a targeted manner, so that the antigen is dissociated. However, almost all of the desorbents cause more or less destruction of the structure of the protein, resulting in denaturation of the protein, resulting in destruction of the structure thereof, and thus cannot be detected. At present, the existing desorption formulas have no good desorption effect on CpG ODN composite adjuvant adsorption type vaccines.
Therefore, a desorption method for quantitatively detecting the effective components of the CpG ODN composite adjuvant adsorption type vaccine is developed, so that a stable solution system is formed, the corresponding antigenic determinant on the surface of the antigen is exposed again, meanwhile, the structural specificity of the antigenic epitope is not influenced, and the method is of great importance for realizing accurate quantitative detection of the antigen in the vaccine. At present, no related report aiming at the dissociation method of the effective components of the CpG ODN composite adjuvant adsorption type vaccine exists at home and abroad.
The dissociation method of the antigen in the CN111812313A aluminum adjuvant adsorption type novel coronavirus fire-extinguishing vaccine adopts a desorption composition comprising 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 dissociation method of the antigen in the aluminum adjuvant adsorption type novel coronavirus inactivated vaccine is not applicable to the CpG ODN composite adjuvant adsorption type vaccine and can only be used for desorbing the single adjuvant, namely the aluminum adjuvant novel coronavirus inactivated vaccine. The desorption process requires ultrasound, which destroys the antigen structure specificity of the CpG ODN composite adjuvant adsorbed vaccine.
CN111840540A A hepatitis B vaccine analytic method, provides a hepatitis B vaccine analytic method, including the following steps: (a) repeatedly freezing and thawing a hepatitis B vaccine sample at a temperature below-15 ℃ for at least one time; (b) mixing a hepatitis B vaccine sample subjected to freeze thawing treatment with hydrochloric acid, and incubating for 1-3h at 37 ℃, wherein the volume ratio of the hepatitis B vaccine sample to the hydrochloric acid is 1: 1-2; (c) and finally adding a protective agent Triton100, and carrying out water bath for 1-3h at 37 ℃, thus completing the resolution. The method can effectively separate the effective components from the adjuvant in the hepatitis B vaccine, so that the effective components can be accurately monitored by the immunoassay kit. Because the prior analytic solution has low resolution ratio, the existing method for determining the activity of the hepatitis B vaccine is an animal method, and the new analytic method can effectively replace animal experiments and save the animal cost. However, the desorption method is suitable for the hepatitis B vaccine adsorbed by the aluminum hydroxide adjuvant, and is not suitable for CpG ODN composite adjuvant adsorption type vaccines. And this method is time consuming.
At present, no method for desorbing the CpG ODN composite adjuvant adsorption type vaccine is reported.
Disclosure of Invention
The present invention has been made to solve the above problems.
In one aspect, the invention provides a desorption composition for an adsorption-type vaccine containing a CpG ODN adjuvant, wherein the desorption composition comprises potassium phosphate salt, M199 culture medium, magnesium chloride and Triton-100.
According to a specific embodiment of the present invention, wherein the potassium phosphate salt is a combination of dipotassium hydrogen phosphate and potassium dihydrogen phosphate;
wherein the molar ratio of the dipotassium hydrogen phosphate to the potassium dihydrogen phosphate is 1-3: 1.
According to a specific embodiment of the present invention, when the desorption composition is present as an aqueous solution as the desorption agent, the final molar concentration of potassium phosphate in the desorption agent is 0.6M to 2.0M, the final molar concentration of magnesium chloride is 0.1M to 0.8M, and the final volume concentration of the M199 culture medium is 10% to 60%; the final volume concentration of Triton-100 in the desorbing agent is 0.1% -3%.
According to a particular embodiment of the invention, when the desorption composition is present as an aqueous solution as desorbing agent, the final molarity of the potassium phosphate salt in the desorbing agent is 0.6M to 1.2M, the final molarity of the magnesium chloride is 0.1M to 0.4M, the final volume concentration of the M199 medium is 20% to 50%, and the final volume concentration of Triton-100 in the desorbing agent is 0.5% to 1%.
According to a specific embodiment of the present invention, the desorption composition of the present invention can be used in the potency determination process of the CPG ODN adjuvant novel coronavirus inactivated vaccine; in the dissociation process, the M199 culture medium and the magnesium chloride are added to play a role in stabilizing the stability of the novel coronavirus inactivated vaccine of the CPG ODN adjuvant, and the antigen bioactivity can be effectively protected.
In another aspect, the present invention provides a kit for an adsorptive vaccine comprising a CpG ODN adjuvant, comprising the above-described desorbing composition.
According to a specific embodiment of the invention, the kit further comprises a general reagent for detecting the effectiveness of the vaccine by a double-antibody sandwich ELISA method and a reaction buffer.
In another aspect, the present invention provides a method for desorbing an adsorbed vaccine containing a CpG ODN adjuvant, the method comprising:
preparing a desorption reagent;
mixing the reference substance and sample with desorption reagent at volume ratio of 1 (1-5), incubating at 4-28 deg.C for 20-120min, and reversing and mixing once every 10-20min during incubation;
centrifuging the obtained mixture at 7000rpm for 10-60 min at 4-28 deg.C to obtain supernatant.
In another aspect, the present invention provides a method for in vitro testing the efficacy of an adsorbed vaccine containing a CpG ODN adjuvant, comprising the steps of:
step 1: preparing the desorption reagent; preferably, 2M potassium phosphate buffer solution and 1M magnesium chloride solution are prepared, then the buffer solution and the M199 culture medium are mixed, water is added to complement the volume, the final molar concentration of potassium phosphate is 0.6M-1.2M, the final molar concentration of magnesium chloride is 0.1M-0.4M, and the final volume concentration of the M199 culture medium is 20% -50%; preferably, Triton-100 is added into the mixed solution, and the final volume concentration of Triton-100 is 0.5% -1%;
step 2: CpG ODN desorption; mixing the reference substance and sample with desorption reagent at volume ratio of 1 (1-5), incubating at 4-28 deg.C for 20-120min, and mixing uniformly by reversing every 10-20min during desorption;
centrifuging the obtained mixture, wherein the centrifugation program is 2000-7000rpm, the centrifugation time is 10-60 min, and the centrifugation temperature is 4-28 ℃ to obtain a supernatant;
and step 3: detecting the in vitro antigen efficacy after desorption; and (3) diluting the supernatant obtained in the step 2, and detecting the OD value of the sample by using a double-antibody sandwich ELISA method.
According to a specific embodiment of the present invention, the step of detecting the OD value of the sample by the double antibody sandwich ELISA method comprises:
and 4, step 4: coating a first antibody specific to an antigen to be detected in a 96-hole microporous plate to prepare a solid phase carrier, and adding a standard substance and a sample to be detected into the micropores, wherein the antigen to be detected is combined with the first antibody on the solid phase carrier;
and 5: and adding a detection antibody specifically bound with the antigen to form a double-antibody sandwich structure, wherein the double-antibody sandwich structure is as follows: a first antibody-vaccine antigen-detection antibody coating the solid phase carrier;
step 6: specifically binding a secondary antibody labeled with HRP to a detection antibody to form a detection complex having the structure: primary antibody-vaccine antigen-detection antibody-HRP labeled secondary antibody coated solid phase carrier;
and 7: adding a chromogenic substrate TMB, reacting with the detection compound obtained in the step 6, and then developing;
and 8: and measuring the light absorption value by a microplate reader. The results of the reference and sample were analyzed using statistical software and the relative potency of the samples in vitro was calculated using the double parallel line method.
In another aspect, the invention provides the use of the composition, or the kit, or the method for the desorption and in vitro potency assay of an adsorptive vaccine containing a CpG ODN adjuvant;
preferably, the CpG ODN adjuvant-containing adsorption type vaccine is a CpG ODN composite adjuvant adsorption type vaccine;
more preferably, the CpG ODN composite adjuvant adsorption type vaccine is a CPG ODN composite adjuvant hepatitis B vaccine and a CPG ODN composite adjuvant combined vaccine;
further preferably, the CpG ODN composite adjuvant adsorption type vaccine is a CPG ODN composite adjuvant novel coronavirus vaccine.
The invention has the beneficial effects that:
the invention provides a desorption composition for an adsorption type vaccine containing a CpG ODN adjuvant, which can dissociate antigens in the adsorption type vaccine containing the CpG ODN adjuvant, and can realize the stabilization of a solution system and the protection of the immune activity of the antigens in the process so as to quickly separate the antigens from an adjuvant compound;
the invention provides a kit for an adsorption type vaccine containing a CpG ODN adjuvant;
when the composition or the kit provided by the invention is used for in-vitro detection of the efficacy of the adsorption type vaccine containing the CpG ODN adjuvant, the detection efficiency is extremely high, and the popularization and the application are convenient.
The invention provides a method for in vitro detecting the effectiveness of an adsorption type vaccine containing a CpG ODN adjuvant, which applies the desorption composition to form a stable solution system, exposes corresponding antigenic determinants on the surface of an antigen again, does not influence the structural specificity of an antigenic epitope, can rapidly dissociate the antigen and keep complete biological deconstruction, and can be directly used for the quantitative detection of the antigen by an enzyme-linked immunosorbent assay. The invention provides a method for desorbing the CpG ODN composite adjuvant adsorption type vaccine for the first time, fills the blank in the aspect of determining the efficacy of the adsorption type vaccine containing the CpG ODN adjuvant, and saves the detection time of the vaccine efficacy.
Drawings
FIG. 1 is the results of example 1 using two parallel lines.
FIG. 2 is the results of the sample sets 1-5 double parallel lines of example 2.
FIG. 3 is the results of the two parallel lines of sample sets 1-6 of example 3.
FIG. 4 is the results of the two parallel lines of sample sets 1-5 of example 5.
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.
The experimental procedures in the following examples are, unless otherwise specified, either conventional or according to the conditions and procedures recommended by the manufacturer. The test materials used in the following examples were purchased from a conventional reagent store unless otherwise specified.
The specific operation of detecting the OD value of the sample by the double-antibody sandwich ELISA method is as follows:
adding a sample: taking an enzyme label plate, and coating a first antibody specifically aiming at the antigen to be detected according to a conventional method in the field. Adding the diluted reference product and sample into an ELISA plate coated with a first antibody specific to the antigen to be detected, wherein each hole is 100 mu L, the dilution of the reference product and the sample is parallel to 2 holes, when the finished product detection is carried out, a negative control parallel 8 holes are needed to be added (the sample adding mode refers to table 1), after gentle shaking and uniform mixing, placing the ELISA plate into a clean sealing bag or sealing the ELISA plate by a sealing plate film, and placing the ELISA plate in a 37 ℃ constant temperature water bath box for incubation for 60-120 min.
TABLE 1
Washing the plate: and taking out the enzyme label plate, washing for 5 times by using a plate washing machine, and then drying by beating.
Adding a detection antibody working solution: the working solution for detection antibody was added to the microplate by using a multi-channel pipette, and 100. mu.L of the working solution was added to each well. Placing the enzyme label plate into a clean sealing bag or sealing with a sealing plate film, and incubating in a 37 deg.C constant temperature water bath for 30-60 min.
Washing the plate: and taking out the enzyme label plate, washing for 5 times by using a plate washing machine, and then drying by beating.
Adding enzyme standard antibody working solution (HRP-labeled secondary antibody): the enzyme-labeled antibody working solution was added to the microplate by a multi-channel pipette at 100. mu.L per well. Placing the enzyme label plate into a clean sealing bag or sealing with a sealing plate film, and incubating in a 37 deg.C constant temperature water bath for 30-60 min.
Sixthly, plate washing: and taking out the enzyme label plate, washing for 5 times by using a plate washing machine, and then drying by beating.
And (c) color development of TMB: sequentially adding the TMB color development A liquid and the TMB color development B liquid into the ELISA plate by a multi-channel pipette, wherein each hole is 50 mu L, gently shaking and uniformly mixing, putting the ELISA plate into a clean sealing bag or sealing the ELISA plate with a sealing plate film, and incubating for 15 minutes in a 37 ℃ constant temperature water bath box.
And stopping reaction: stop solution was added to the microplate by a multichannel pipettor, 50. mu.L per well.
Ninthly, determination by an enzyme labeling instrument: the OD value of each well was measured by reading with a microplate reader at 450/630nm dual wavelength.
Example 1
The implementation provides a method for detecting antigen efficacy in a CpG ODN composite adjuvant adsorption type vaccine, which comprises the following specific steps:
step 1: preparing a desorption reagent: wherein the final molarity of the potassium phosphate salt is 0.6M, the final molarity of the magnesium chloride is 0.1M, the final volume concentration of the Triton-100 is 1%, and the final volume concentration of the M199 culture medium is 20%;
step 2: desorbing the CpG ODN composite adjuvant adsorption type vaccine reference substance and the sample to be detected: mixing the reference substance and sample with desorption reagent at volume ratio of 1:1, incubating at 28 deg.C for 60min, and mixing uniformly by reversing every 20min during incubation;
centrifuging the obtained mixture at 6000rpm for 20min at 4 deg.C to obtain supernatant;
and 3, step 3: detecting the in vitro antigen efficacy after desorption; diluting the supernatant CpG ODN composite adjuvant adsorption type vaccine desorbed in the step 2 according to 2X, 4X, 8X and 16X, detecting the OD value of a sample by adopting a double-antibody sandwich ELISA method, carrying out in-vitro relative efficacy statistical analysis on the result by adopting the basic principle of a biological detection double-parallel line method, and calculating the efficacy of the vaccine to be detected relative to a reference product, wherein the detection result is shown in a table 2.
TABLE 2
Statistical results of the in vitro relative potency obtained with this method are shown in table 3.
TABLE 3
The results obtained for the two parallel lines are shown in figure 1.
Example 2
The implementation provides antigen dissociation results in different batches of CpG ODN composite adjuvant adsorption type novel coronavirus inactivated vaccines, and the applicability of the dissociation method to different batches of samples is investigated.
The specific desorption and antigen potency assay was the same as in example 1.
The detection results of the antigen efficacy in different batches of CpG ODN composite adjuvant adsorption type novel coronavirus inactivated vaccines are shown in Table 4.
TABLE 4
The results for samples 1-5 are shown in FIG. 2 as doubly parallel lines.
From table 4, the invention has good applicability to CpG ODN composite adjuvant adsorption type novel coronavirus inactivated vaccines.
Example 3
This example verifies the desorption applicability of the CpG ODN composite adjuvant adsorption type novel inactivated vaccine using desorption compositions of different component ratios, different desorption conditions, and different centrifugation parameters.
The sample to be tested used in this example had a theoretical antigen concentration of 13U/ml and the sample dilution protocol was 2X, 4X, 8X, 16X. Antigen potency tests were all considered eligible between 0.5 and 2.0.
The desorption conditions and antigen potency assay results for each sample set are shown in table 5.
The procedure for the specific antigen potency assay was the same as in example 1.
TABLE 5
The results for samples 1-6 are shown in figure 3 for the double parallel lines.
As shown in Table 5, the detection results are all qualified, and the effectiveness of the desorption composition and the desorption reagent for the CpG ODN adjuvant-containing adsorptive vaccine and the method for detecting the effectiveness of the CpG ODN adjuvant-containing adsorptive vaccine in vitro are verified.
Example 4
This example evaluates the interference of the desorbing composition in desorbing the CpG ODN composite adjuvant adsorptive vaccine.
The OD values of the desorbing composition at 2X, 4X, 8X, 16X dilution were determined and compared to the negative control.
Evaluation criteria: the OD values of all dilution concentration groups of the desorption composition are not different from the negative control OD value, so that the CpG ODN composite adjuvant adsorption type vaccine desorption reagent in the method is proved to have no interference to detection; and calculating the average value of OD values of the negative control detection results of 8 times, and marking as S (when the S value is less than 0.05, the S value is counted by 0.05), wherein the absorbance value of the reference substance is more than or equal to 2.1 times of the S, namely the negative control result is positive, otherwise, the negative control result is negative.
TABLE 6
The evaluation results are shown in table 6, and as can be seen from table 6, the OD values of all dilution concentration groups of the desorption composition have no obvious difference from the negative control OD value, which proves that the CpG ODN composite adjuvant adsorption type vaccine desorption reagent in the method has no interference to detection. This example demonstrates that knowledge of the adsorption composition does not interfere with the desorption of the CpG ODN co-adjuvant adsorption type novel inactivated vaccine.
Comparative example 1
This comparative example uses prior art methods to test the efficacy of an adsorbed vaccine containing a CpG ODN adjuvant.
The theoretical antigen concentration of the sample to be detected used in the comparative example is 13U/ml, and the sample dilution scheme is 2X, 4X, 8X and 16X. Antigen potency tests were all considered eligible between 0.5 and 2.0. The antigen potency was determined as in example 1. The desorption conditions and antigen potency assay results for each sample set are shown in table 7.
TABLE 7
As can be seen from table 7, group 5 shows that acceptable test results can be obtained with the desorption composition and method of the present invention; the detection results of other groups 4 except the group 5 are not qualified, which indicates that other desorption compositions are not suitable for the CpG ODN composite adjuvant adsorption type vaccine.
The results for sample sets 1-5 were shown in FIG. 4 as doubly parallel lines.
The above results indicate that other desorbing compositions are not suitable for CpG ODN composite adjuvant adsorption type vaccines, but the present invention can perform good desorption on CpG ODN composite adjuvant adsorption type vaccines.
Claims (10)
1. A desorption composition of an adsorption type vaccine containing CpG ODN, which is characterized by comprising potassium phosphate salt, M199 culture medium, magnesium chloride and Triton-100.
2. The desorption composition of claim 1, wherein the potassium phosphate salt is a combination of dipotassium hydrogen phosphate and potassium dihydrogen phosphate;
wherein the molar ratio of the dipotassium hydrogen phosphate to the potassium dihydrogen phosphate is 1-3: 1.
3. Desorption composition according to claim 1 or 2, wherein the final molar concentration of potassium phosphate in the desorption agent is 0.6M to 2.0M, the final molar concentration of magnesium chloride is 0.1M to 0.8M, and the final volume concentration of M199 medium is 10% to 60% when the desorption composition is present as an aqueous solution as desorption agent; the final volume concentration of Triton-100 in the desorbing agent is 0.1% -3%.
4. The desorption composition as claimed in claim 3, wherein the final molarity of potassium phosphate salt in the desorption agent is 0.6M-1.2M, the final molarity of magnesium chloride is 0.1M-0.4M, and the final volume concentration of M199 culture medium is 20% -50%; the final volume concentration of Triton-100 in the desorbing agent is 0.5% -1%.
5. A kit for use in an adsorptive vaccine comprising a CpG ODN adjuvant, comprising the desorbing composition of any one of claims 1-4.
6. The kit according to claim 5, further comprising a general reagent for detecting the efficacy of the vaccine by a double antibody sandwich ELISA method and a reaction buffer.
7. A method for desorbing an adsorbed vaccine containing a CpG ODN adjuvant, the method comprising:
formulating the desorbing agent of claim 3 or 4;
mixing the reference substance and sample with desorption reagent at volume ratio of 1 (1-5), incubating at 4-28 deg.C for 20-120min, and reversing and mixing once every 10-20min during incubation;
centrifuging the obtained mixture at 7000rpm for 10-60 min at 4-28 deg.C to obtain supernatant.
8. A method for in vitro testing the efficacy of an adsorbed vaccine containing a CpG ODN adjuvant, the method comprising the steps of:
step 1: formulating the desorbing agent of claim 3 or 4;
step 2: CpG ODN desorption; mixing the reference substance and sample with desorption reagent at volume ratio of 1 (1-5), incubating at 4-28 deg.C for 20-120min, and reversing and mixing once every 10-20min during incubation;
centrifuging the obtained mixture, wherein the centrifugation program is 2000-7000rpm, the centrifugation time is 10-60 min, and the centrifugation temperature is 4-28 ℃ to obtain a supernatant;
and step 3: detecting the in vitro antigen efficacy after desorption; and (3) diluting the supernatant obtained in the step 2, and detecting the OD value of the sample by using a double-antibody sandwich ELISA method.
9. The method of claim 8, wherein the step of detecting the OD of the sample by the double antibody sandwich ELISA method comprises:
and 4, step 4: coating a first antibody specific to an antigen to be detected in a 96-hole microporous plate to prepare a solid phase carrier, and adding a standard substance and a sample to be detected into the micropores, wherein the antigen to be detected is combined with the first antibody on the solid phase carrier;
and 5: and adding a detection antibody specifically combined with the antigen to form a double-antibody sandwich structure, wherein the double-antibody sandwich structure is as follows: a first antibody-vaccine antigen-detection antibody coating the solid phase carrier;
step 6: specifically binding a secondary antibody labeled with HRP to a detection antibody to form a detection complex having the structure: primary antibody-vaccine antigen-detection antibody-HRP labeled secondary antibody coated solid phase carrier;
and 7: adding a chromogenic substrate TMB, reacting with the detection compound obtained in the step 6, and then developing;
and 8: measuring a light absorption value by an enzyme-labeling instrument; the results of the reference and sample were analyzed using statistical software and the relative potency of the samples in vitro was calculated using the double parallel line method.
10. Use of the composition of any one of claims 1 to 4, or the kit of claim 5 or 6, or the method of any one of claims 7 to 9, for desorption and/or in vitro potency testing of CpG ODN adjuvant-containing adsorptive vaccines.
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Citations (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040161776A1 (en) * | 2001-10-23 | 2004-08-19 | Maddon Paul J. | PSMA formulations and uses thereof |
| CA2546222A1 (en) * | 2003-11-19 | 2005-06-09 | Chiron Corporation | Methods and reagents for treating, preventing and diagnosing bunyavirus infection |
| US20050153381A1 (en) * | 2002-02-14 | 2005-07-14 | Marusich Michael F. | Immunocapture of mitochondrial protein complexes |
| WO2007108675A1 (en) * | 2006-03-17 | 2007-09-27 | Crossbeta Biosciences B.V. | Methods of binding of cross-beta structures by chaperones |
| CN101745109A (en) * | 2010-02-01 | 2010-06-23 | 云南沃森生物技术股份有限公司 | Method for preparing nasal-spray type influenza virus vaccine containing CpG ODN and poly I:G adjuvant |
| CN101790383A (en) * | 2007-06-29 | 2010-07-28 | 分子免疫中心 | Production of an homogeneous vaccine preparation for cancer treatment |
| CN101971030A (en) * | 2007-12-24 | 2011-02-09 | 诺华有限公司 | Assays for adsorbed influenza vaccines |
| CN102068692A (en) * | 2010-12-30 | 2011-05-25 | 北京民海生物科技有限公司 | Influenza virus splitting vaccine and preparation method thereof |
| CN102221611A (en) * | 2011-04-25 | 2011-10-19 | 河南科技学院 | Duck-origin coronavirus sandwich ELISA kit and its application |
| US20140294826A1 (en) * | 2007-02-20 | 2014-10-02 | Tufts University | Methods and compositions with a recombinant neutralizing binding protein for treating toxin exposure |
| CN104634959A (en) * | 2013-11-08 | 2015-05-20 | 丽珠集团疫苗工程股份有限公司 | Treatment liquid and method using same to measure antigen content of aluminum salt adsorption type vaccines |
| CN105267988A (en) * | 2015-11-18 | 2016-01-27 | 北京中农生物工程有限公司 | Novel echinococosis granulosis vaccine with CPG DNA (deoxyribonucleic acid) immune adjuvants |
| US20160362501A1 (en) * | 2007-02-20 | 2016-12-15 | Tufts University | Methods, compositions and kits for treating a subject using a recombinant heteromultimeric neutralizing binding protein |
| WO2017009873A1 (en) * | 2015-07-16 | 2017-01-19 | Bharat Biotech International Limited | Vaccine compositions |
| CN107281476A (en) * | 2017-04-06 | 2017-10-24 | 中国医科大学 | A kind of Antigenic Peptide RL adjuvants CpGODN7909 conjugates and its preparation method and application |
| US20170340723A1 (en) * | 2014-12-11 | 2017-11-30 | Intervet Inc. | Process for Ready-to-Use PCV2/M.HYO Combination Vaccine |
| WO2018045344A1 (en) * | 2016-09-01 | 2018-03-08 | Takeda Vaccines, Inc. | Methods for producing virus for vaccine production |
| CN108226480A (en) * | 2017-12-22 | 2018-06-29 | 北京民海生物科技有限公司 | The method for measuring D- antigenic contents in aluminium salt absorbent-type DTap-IPV combined vaccines |
| CA3064475A1 (en) * | 2017-05-31 | 2018-12-06 | Texas Tech University System | Methods and devices for the treatment of food allergies |
| CN110433285A (en) * | 2019-06-20 | 2019-11-12 | 杭州师范大学 | A kind of individuation knubble antigen peptide vaccine and preparation method thereof |
| US20190352342A1 (en) * | 2017-01-30 | 2019-11-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Hepatitis e virus orf2 capsid polypeptides and uses thereof |
| CN110917344A (en) * | 2019-12-26 | 2020-03-27 | 北京科兴中维生物技术有限公司 | Liquid vaccine composition and application thereof |
| CN111053898A (en) * | 2019-12-26 | 2020-04-24 | 北京科兴中维生物技术有限公司 | Vaccine composition and application thereof |
| CN111278458A (en) * | 2017-10-25 | 2020-06-12 | 默沙东公司 | Adjuvant vaccines |
| CN111812313A (en) * | 2020-06-22 | 2020-10-23 | 北京生物制品研究所有限责任公司 | Dissociation method of antigen in aluminum adjuvant adsorption type novel coronavirus inactivated vaccine |
| CN112138155A (en) * | 2019-06-28 | 2020-12-29 | 怡道生物科技(苏州)有限公司 | Compound adjuvant system and method for preparing adjuvant |
| CN112379087A (en) * | 2020-10-21 | 2021-02-19 | 中国医学科学院医学生物学研究所 | Lysate applied to novel coronavirus inactivated vaccine and antigen dissociation method |
-
2022
- 2022-05-09 CN CN202210495902.1A patent/CN114594257B/en active Active
Patent Citations (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040161776A1 (en) * | 2001-10-23 | 2004-08-19 | Maddon Paul J. | PSMA formulations and uses thereof |
| US20050153381A1 (en) * | 2002-02-14 | 2005-07-14 | Marusich Michael F. | Immunocapture of mitochondrial protein complexes |
| CA2546222A1 (en) * | 2003-11-19 | 2005-06-09 | Chiron Corporation | Methods and reagents for treating, preventing and diagnosing bunyavirus infection |
| WO2007108675A1 (en) * | 2006-03-17 | 2007-09-27 | Crossbeta Biosciences B.V. | Methods of binding of cross-beta structures by chaperones |
| US20140294826A1 (en) * | 2007-02-20 | 2014-10-02 | Tufts University | Methods and compositions with a recombinant neutralizing binding protein for treating toxin exposure |
| US20160362501A1 (en) * | 2007-02-20 | 2016-12-15 | Tufts University | Methods, compositions and kits for treating a subject using a recombinant heteromultimeric neutralizing binding protein |
| CN101790383A (en) * | 2007-06-29 | 2010-07-28 | 分子免疫中心 | Production of an homogeneous vaccine preparation for cancer treatment |
| CN101971030A (en) * | 2007-12-24 | 2011-02-09 | 诺华有限公司 | Assays for adsorbed influenza vaccines |
| CN101745109A (en) * | 2010-02-01 | 2010-06-23 | 云南沃森生物技术股份有限公司 | Method for preparing nasal-spray type influenza virus vaccine containing CpG ODN and poly I:G adjuvant |
| CN102068692A (en) * | 2010-12-30 | 2011-05-25 | 北京民海生物科技有限公司 | Influenza virus splitting vaccine and preparation method thereof |
| CN102221611A (en) * | 2011-04-25 | 2011-10-19 | 河南科技学院 | Duck-origin coronavirus sandwich ELISA kit and its application |
| CN104634959A (en) * | 2013-11-08 | 2015-05-20 | 丽珠集团疫苗工程股份有限公司 | Treatment liquid and method using same to measure antigen content of aluminum salt adsorption type vaccines |
| US20170340723A1 (en) * | 2014-12-11 | 2017-11-30 | Intervet Inc. | Process for Ready-to-Use PCV2/M.HYO Combination Vaccine |
| WO2017009873A1 (en) * | 2015-07-16 | 2017-01-19 | Bharat Biotech International Limited | Vaccine compositions |
| CN105267988A (en) * | 2015-11-18 | 2016-01-27 | 北京中农生物工程有限公司 | Novel echinococosis granulosis vaccine with CPG DNA (deoxyribonucleic acid) immune adjuvants |
| WO2018045344A1 (en) * | 2016-09-01 | 2018-03-08 | Takeda Vaccines, Inc. | Methods for producing virus for vaccine production |
| US20190352342A1 (en) * | 2017-01-30 | 2019-11-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Hepatitis e virus orf2 capsid polypeptides and uses thereof |
| CN107281476A (en) * | 2017-04-06 | 2017-10-24 | 中国医科大学 | A kind of Antigenic Peptide RL adjuvants CpGODN7909 conjugates and its preparation method and application |
| CA3064475A1 (en) * | 2017-05-31 | 2018-12-06 | Texas Tech University System | Methods and devices for the treatment of food allergies |
| CN111278458A (en) * | 2017-10-25 | 2020-06-12 | 默沙东公司 | Adjuvant vaccines |
| CN108226480A (en) * | 2017-12-22 | 2018-06-29 | 北京民海生物科技有限公司 | The method for measuring D- antigenic contents in aluminium salt absorbent-type DTap-IPV combined vaccines |
| CN110433285A (en) * | 2019-06-20 | 2019-11-12 | 杭州师范大学 | A kind of individuation knubble antigen peptide vaccine and preparation method thereof |
| CN112138155A (en) * | 2019-06-28 | 2020-12-29 | 怡道生物科技(苏州)有限公司 | Compound adjuvant system and method for preparing adjuvant |
| CN110917344A (en) * | 2019-12-26 | 2020-03-27 | 北京科兴中维生物技术有限公司 | Liquid vaccine composition and application thereof |
| CN111053898A (en) * | 2019-12-26 | 2020-04-24 | 北京科兴中维生物技术有限公司 | Vaccine composition and application thereof |
| CN111812313A (en) * | 2020-06-22 | 2020-10-23 | 北京生物制品研究所有限责任公司 | Dissociation method of antigen in aluminum adjuvant adsorption type novel coronavirus inactivated vaccine |
| CN112379087A (en) * | 2020-10-21 | 2021-02-19 | 中国医学科学院医学生物学研究所 | Lysate applied to novel coronavirus inactivated vaccine and antigen dissociation method |
Non-Patent Citations (5)
| Title |
|---|
| WENTING LU 等: "CpG ODN as an adjuvant arouses the vigor of B cells by relieving the negative regulation of surface TLR9 to enhance the antibody response to vaccine", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| YU WENQIAN 等: "Enhanced Immunostimulatory Activity of a Cytosine-Phosphate-Guanosine Immunomodulator by the Assembly of Polymer DNA Wires and Spheres", 《ACS APPLIED MATERIALS & INTERFACES》 * |
| 年悬悬 等: "Toll样受体激动剂佐剂的研究进展", 《中国生物制品学杂志》 * |
| 钱雯 等: "CpG ODN佐剂对乙肝疫苗抗原免疫原性的影响", 《免疫学杂志》 * |
| 颜伟等: "CpG ODN107增强人脑胶质瘤细胞放射敏感性的研究", 《西南国防医药》 * |
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