CN110554188A - Method for detecting content of adjuvant adsorption component vaccine - Google Patents
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- CN110554188A CN110554188A CN201911008908.6A CN201911008908A CN110554188A CN 110554188 A CN110554188 A CN 110554188A CN 201911008908 A CN201911008908 A CN 201911008908A CN 110554188 A CN110554188 A CN 110554188A
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- 229960005486 vaccine Drugs 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 33
- 239000002671 adjuvant Substances 0.000 title claims abstract description 22
- 238000001179 sorption measurement Methods 0.000 title claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 35
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims abstract description 29
- 229920000053 polysorbate 80 Polymers 0.000 claims abstract description 29
- 238000003795 desorption Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 56
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 42
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 42
- 150000004676 glycans Chemical class 0.000 claims description 41
- 229920001282 polysaccharide Polymers 0.000 claims description 41
- 239000005017 polysaccharide Substances 0.000 claims description 41
- 239000012224 working solution Substances 0.000 claims description 16
- 229940031999 pneumococcal conjugate vaccine Drugs 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 9
- 239000003085 diluting agent Substances 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 3
- 238000010998 test method Methods 0.000 claims description 3
- 230000003113 alkalizing effect Effects 0.000 claims description 2
- 230000003472 neutralizing effect Effects 0.000 claims description 2
- 239000003381 stabilizer Substances 0.000 claims description 2
- 229940124931 vaccine adjuvant Drugs 0.000 claims description 2
- 229940124856 vaccine component Drugs 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 238000011088 calibration curve Methods 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 4
- 238000004879 turbidimetry Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- 108010060123 Conjugate Vaccines Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229940031670 conjugate vaccine Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 229940031348 multivalent vaccine Drugs 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 229940031937 polysaccharide vaccine Drugs 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 2
- 239000001384 succinic acid Substances 0.000 description 2
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 229940021995 DNA vaccine Drugs 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- XHMJOUIAFHJHBW-CBPJZXOFSA-N [(2r,3s,4r,5s)-5-amino-3,4,6-trihydroxyoxan-2-yl]methyl dihydrogen phosphate Chemical compound N[C@@H]1C(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O XHMJOUIAFHJHBW-CBPJZXOFSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000000240 adjuvant effect Effects 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 229940103272 aluminum potassium sulfate Drugs 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940126580 vector vaccine Drugs 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56944—Streptococcus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Abstract
The invention belongs to the technical field of biology, and relates to a method for detecting the content of an adjuvant adsorption component vaccine. The invention solves the problem of low sensitivity of the existing adjuvant adsorption component vaccine content detection method. The detection method comprises the steps of adding Tween-80 solution into adjuvant adsorption component vaccine, removing adjuvant influence through desorption treatment, and determining the content of each component of the vaccine through a standard curve method. The method has the characteristics of high detection sensitivity and good repeatability.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting the content of an adjuvant adsorption component vaccine.
Background
in the quality control of the component vaccine, the quantification of each component of the vaccine is one of important quality control items. At present, the quantitative detection method of each component in the component vaccine comprises the following steps:
Chemical color development method:
some components in the vaccine have light absorption in a specific wavelength range, or form a substance with light absorption after chemical treatment, and the light absorption degree and the content have a correlation relationship, so that the vaccine can be used for content detection. For example, sialic acid is one of the active ingredients in group C meningococcal polysaccharide conjugate vaccine, and the maximum absorbance at 585nm is obtained by extracting sialic acid from group C meningococcal polysaccharide with resorcinol. The limitation of this method is that most multivalent vaccines have similar chemical properties of each component, making it difficult to find a chemical treatment for one of the components.
Chromatographic analysis:
Anion chromatography is used for analysis of various monosaccharides, disaccharides, oligosaccharides, polysaccharides. For example, group A polysaccharide in group A and group C meningococcal polysaccharide vaccines is hydrolyzed by acid to produce 6-phosphomannosamine monomer, and the content is measured by high performance anion exchange chromatography-pulsed amperometric detection. The limitations of this method are that it is difficult to achieve baseline separation of components in multivalent vaccines, the chromatographic conditions are complex, and the requirements for reagents and the environment are high.
Enzyme-linked immunosorbent assay:
Double antibody sandwich ELISA is commonly used for the quantitative detection of antigens. A. C, W135 and Y group tetravalent meningococcus polysaccharide vaccine, and a double-antibody sandwich quantitative ELISA method is adopted for detecting the content of each serogroup polysaccharide protein conjugate. This method requires the preparation of each population of polysaccharide-specific monoclonal antibodies as capture antibodies. The monoclonal antibody has higher preparation cost and lower yield, and is difficult to adapt to the requirement of large-scale detection.
Immune rocket electrophoresis method:
The immune rocket electrophoresis can preliminarily calculate the polysaccharide content by performing linear regression analysis on the migration rate of the polysaccharide with different concentrations to be detected according to the migration rate of the polysaccharide with known concentration in the same gel. The method has complex operation process and is a method for roughly measuring the content of the polysaccharide.
Rate turbidimetry:
The rate turbidimetry is an immunochemical method for detecting the content of each component of the component vaccine. In the method, under the condition that the antibody exists in excess, the rate of forming the complex aggregate by the antigen and the antibody is in a certain proportional relation with the amount of the antigen existing in the system. The content of a certain antigen in the mixture can be detected by utilizing the principle. The speed turbidimetry is simple, convenient and quick to operate, high in automation degree, different antigens can be detected by adopting the same parameter setting, and the method can meet the requirement of large-scale detection.
the content of the adjuvant adsorption component vaccine is detected by adopting a rate turbidimetry method, and the currently general method is to directly carry out desorption treatment and then determine the content of each component. The adjuvant has less adsorbed vaccine antigen amount, so that the sensitivity requirement on the detection method is higher.
Therefore, in order to solve the technical problems in the existing method, the invention provides a brand-new detection method.
Disclosure of Invention
the invention aims to provide a method for detecting the content of an adjuvant adsorption component vaccine.
the method can effectively solve the problems of low sensitivity and large fluctuation of detection results of the existing adjuvant adsorption component vaccine content detection method.
in order to achieve the above purpose, the strategy adopted by the invention is as follows: adding Tween-80 solution into adjuvant adsorption component vaccine, desorbing to eliminate adjuvant effect, and measuring the content of each component of the vaccine by standard curve method.
The detection method comprises the following steps:
the method comprises the following steps: adding Tween-80 solution into adjuvant adsorption vaccine;
Step two: desorbing the vaccine component and adjuvant with alkali, and neutralizing with acid;
step three: preparing a calibration working solution from each component in the vaccine independently;
Step four: the calibration working solution is treated in the same way in the first step and the second step;
Step five: diluting the treated calibration working solution into a series of concentration gradients as a standard curve;
Step six: reacting the standard curve with a test reagent, and drawing the standard curve by taking the content of each component as an abscissa and the response value as an ordinate;
Step seven: the vaccine reacts with the test reagent, and the content of each component of the vaccine is determined by a standard curve.
Preferably, in the first step, the tween-80 solution is added to the vaccine in a volume of no more than 1% of the total volume.
preferably, in step one, tween-80 is added to the vaccine at a final concentration of no more than 1%, preferably 0.01%.
Preferably, in the fourth step, the volume of the tween-80 solution added into the calibration working solution is not more than 1% of the total volume.
preferably, in the fourth step, tween-80 is added into the calibration working solution to a final concentration of not more than 1%, preferably 0.01%.
wherein, in the step one, the adjuvant adsorption vaccine is: DNA vaccines adsorbed by aluminum phosphate, aluminum hydroxide or aluminum potassium sulfate, component vaccines, conjugate vaccines, combined vaccines, recombinant vaccines, virus vector vaccines and the like.
Wherein, in the second step, the alkali is sodium hydroxide solution;
the acid is: citric acid;
In the sixth step, the test reagent is: serum or antibodies matched to vaccine components;
Most preferably, the detection method of the present invention comprises the steps of:
(1) Taking 495 mu L of pneumococcus conjugate, sequentially adding 5 mu L of 1% Tween-80 solution, 15 mu L of 1mol/L sodium hydroxide solution and 6 mu L of 1mol/L citric acid solution, fully and uniformly mixing in each step, wherein the polysaccharide concentration is 20 mu g/ml, and taking the polysaccharide as a calibration substance working solution;
(2) Diluting the working solution of the calibration standard into a series of concentration gradient solutions with polysaccharide concentration of 5.5 mug/ml, 4.5 mug/ml, 3.5 mug/ml and 2.5 mug/ml by using a diluent as a standard curve;
(3) Reacting the standard curve with serum matched with the conjugate, and drawing the standard curve by taking the polysaccharide content as an abscissa and the response value as an ordinate;
(4)
and taking 500 mu L of pneumococcal conjugate vaccine, sequentially adding 5 mu L of 1% Tween-80 solution, 15 mu L of 1mol/L sodium hydroxide solution and 6 mu L of 1mol/L citric acid solution, fully and uniformly mixing in each step, then reacting with serum, and determining the content of each component of the vaccine through a standard curve.
most preferably, the detection method of the present invention comprises the steps of:
(1) Taking 495 mul of pneumococcus conjugate, sequentially adding 5 mul of 1% Tween-80 solution, 15 mul of 1mol/L sodium hydroxide solution and 6 mul of 1mol/L citric acid solution, and fully and uniformly mixing in each step, wherein the polysaccharide concentration is 20 mug/ml;
(2) diluting the conjugate with diluent until polysaccharide concentration is 4.2 μ g/ml;
(3) Taking 500 mu L of pneumococcal conjugate vaccine, sequentially adding 5 mu L of 1% Tween-80 solution, 15 mu L of 1mol/L sodium hydroxide solution and 6 mu L of 1mol/L citric acid solution, and fully and uniformly mixing in each step, wherein the concentration of each type of conjugate polysaccharide in the vaccine is 4.2 mu g/ml;
(4) The conjugate and vaccine were simultaneously reacted with serum and the response values (ratio of vaccine response value to conjugate response value) of both were compared.
The method can effectively solve the problems existing in the existing method, and effectively improve the problems of low sensitivity and large fluctuation of detection results of the adjuvant adsorption component vaccine content detection method. The invention improves the accuracy, stability and precision of the detection result, shortens the detection time and reduces the cost.
Drawings
FIG. 1 is a graph comparing standard curves for pneumococcal conjugate type 1 in a pneumococcal conjugate vaccine of example 1 of the present invention;
FIG. 2 is a comparison of standard curves for pneumococcal conjugate type 6B in the pneumococcal conjugate vaccine of example 2 of the present invention;
FIG. 3 is a comparison of standard curves for pneumococcal conjugate type 12F in the pneumococcal conjugate vaccine of example 3 of the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to be limiting.
example 1 detection of pneumococcal polysaccharide type 1 in pneumococcal conjugate vaccine
1. 495 ul of pneumococcal conjugate type 1 is taken, 5 ul of 1% Tween-80 solution, 15 ul of 1mol/L sodium hydroxide solution and 6 ul of 1mol/L citric acid solution are sequentially added, and each step is fully and uniformly mixed. The polysaccharide concentration was 20. mu.g/ml, which was used as the calibration sample working solution 1.
2. The calibration sample solution 1 was diluted with a diluent to a series of concentration gradient solutions of 5.5. mu.g/ml, 4.5. mu.g/ml, 3.5. mu.g/ml and 2.5. mu.g/ml of polysaccharide as a calibration curve 1.
3. 500 mu L of type 1 pneumococcal conjugate is taken, 15 mu L of 1mol/L sodium hydroxide solution and 6 mu L of 1mol/L citric acid solution are sequentially added, and each step is fully and uniformly mixed. The polysaccharide concentration was 20. mu.g/ml, which was used as the calibration sample solution 2.
4. The calibration sample solution 2 was diluted with a diluent to a series of concentration gradient solutions having a polysaccharide concentration of 5.5. mu.g/ml, 4.5. mu.g/ml, 3.5. mu.g/ml and 2.5. mu.g/ml, and used as the calibration curve 2.
5. And taking 500 mu L of pneumococcal conjugate vaccine, and sequentially adding 5 mu L of 1% Tween-80 solution, 15 mu L of 1mol/L sodium hydroxide solution and 6 mu L of 1mol/L citric acid solution, and fully and uniformly mixing in each step.
6. FIG. 1 is a graph comparing standard curves for pneumococcal conjugate type 1.
7. As can be seen from the figure, the standard curve 1: r0.9986, and y 7.174 x-4.1335; standard curve 2: r0.9976, and y 2.6745x + 5.4007.
8. table 1 shows the comparison of the slopes of the two standard curves and the comparison of the results of the detection of the polysaccharide content in the vaccine.
9. As can be seen from the table, the slope of the standard curve 1 is significantly higher than that of the standard curve 2, indicating that the sensitivity of the standard curve 1 is higher. The results of detecting the polysaccharide content in the vaccine by the two standard curves both meet the detection standard.
TABLE 1
Example 2 detection of type 6B pneumococcal polysaccharide content in pneumococcal conjugate vaccine
1. 495 ul of 6B type pneumococcal conjugate is taken, 5 ul of 1% Tween-80 solution, 15 ul of 1mol/L sodium hydroxide solution and 6 ul of 1mol/L citric acid solution are sequentially added, and each step is fully and uniformly mixed. The polysaccharide concentration was 40. mu.g/ml, which was used as the calibration sample working solution 1.
2. the working solution 1 was diluted with a diluent to a series of concentration gradient solutions of 11. mu.g/ml, 9. mu.g/ml, 7. mu.g/ml and 5. mu.g/ml polysaccharide concentration as the calibration curve 1.
3. 500 mul of 6B pneumococcal conjugate is taken, 15 mul of 1mol/L sodium hydroxide solution and 6 mul of 1mol/L citric acid solution are sequentially added, and each step is fully and uniformly mixed. The polysaccharide concentration was 40. mu.g/ml, which was used as the calibration sample solution 2.
4. the working solution 2 was diluted with a diluent to a series of concentration gradient solutions of 11. mu.g/ml, 9. mu.g/ml, 7. mu.g/ml and 5. mu.g/ml polysaccharide concentration as the calibration curve 2.
5. The experimental procedure was the same as in example 1, Steps 5-6.
6. FIG. 2 is a comparison of standard curves for pneumococcal conjugate type 6B.
7. As can be seen from the figure, the standard curve 1: r0.9968, and y 7.5788 x-24.164; standard curve 2: r0.9861, and y 1.0305 x-3.5215.
8. table 1 shows the comparison of the slopes of the two standard curves and the comparison of the results of the detection of the polysaccharide content in the vaccine.
9. As can be seen from the table, the slope of the standard curve 1 is significantly higher than that of the standard curve 2, indicating that the sensitivity of the standard curve 1 is higher. The results of detecting the polysaccharide content in the vaccine by the two standard curves both meet the detection standard.
example 3 detection of the amount of pneumococcal polysaccharide type 12F in pneumococcal conjugate vaccines
1. The experimental procedure was the same as in example 1, Steps 1-6.
2. FIG. 2 is a graph comparing standard curves for pneumococcal conjugate type 12F.
3. As can be seen from the figure, the standard curve 1: r is 0.9979, and the linear regression equation is y is 4.4405 x-0.0833; standard curve 2: r is 0.9832 and the linear regression equation is y 2.9325x + 2.7837.
4. Table 1 shows the comparison of the slopes of the two standard curves and the comparison of the results of the detection of the polysaccharide content in the vaccine.
As can be seen from the table, the slope of the standard curve 1 is significantly higher than that of the standard curve 2, indicating that the sensitivity of the standard curve 1 is higher. The results of detecting the polysaccharide content in the vaccine by the two standard curves both meet the detection standard.
Example 4 screening experiment
The core point of the invention is that the Tween-80 solution is added into the adjuvant adsorption component vaccine.
1. In the screening process, tween solution is added into a buffer system, and the effect of tween in the buffer system is not obvious. After changing the protocol, tween solution was added to the vaccine and showed significant optimization as in examples 1-3. Finally, the Tween-80 solution is preferably added into the adjuvant adsorption component vaccine.
2. In the screening process, tween-80 and succinic acid are mixed and then added into the vaccine as a stabilizer, and the addition of succinic acid is found to have no further optimization effect on the test method. Based on the principle of simplifying the reaction system, the Tween-80 solution is finally preferred.
3. During the screening process, the final concentration of tween-80 was also screened.
(1) Taking 495 mul of pneumococcus conjugate, sequentially adding 5 mul of 1% Tween-80 solution, 15 mul of 1mol/L sodium hydroxide solution and 6 mul of 1mol/L citric acid solution, and fully and uniformly mixing in each step, wherein the polysaccharide concentration is 20 mug/ml;
(2) Diluting the conjugate with diluent until polysaccharide concentration is 4.2 μ g/ml;
(3) Taking 500 mu L of pneumococcal conjugate vaccine, sequentially adding 5 mu L of 1% Tween-80 solution, 15 mu L of 1mol/L sodium hydroxide solution and 6 mu L of 1mol/L citric acid solution, and fully and uniformly mixing in each step, wherein the concentration of each type of conjugate polysaccharide in the vaccine is 4.2 mu g/ml;
(4) The conjugate and vaccine were simultaneously reacted with serum and the response values (ratio of vaccine response value to conjugate response value) of both were compared.
table 2 shows the results of comparing the response values of the vaccines and the conjugates at the same concentration after adding different concentrations of Tween-80 to the vaccines.
TABLE 2
By comparison, the final preferred final concentration is the lowest 0.01% Tween-80 solution.
Claims (7)
1. A method for detecting the content of adjuvant adsorbed component vaccine is characterized in that Tween-80 is added into the vaccine as a stabilizer, the influence of the adjuvant is eliminated through desorption treatment, and the content of each component of the vaccine is measured through a standard curve method.
2. the detection method according to claim 1, characterized by comprising the steps of:
The method comprises the following steps: adding Tween-80 solution into adjuvant adsorption vaccine;
step two: desorbing the vaccine component and adjuvant with alkali, and neutralizing with acid;
Step three: preparing a calibration working solution from each component in the vaccine independently;
Step four: the calibration working solution is treated in the same way in the first step and the second step;
Step five: diluting the treated calibration working solution into a series of concentration gradients as a standard curve;
step six: reacting the standard curve with a test reagent, and drawing the standard curve by taking the content of each component as an abscissa and the response value as an ordinate;
step seven: the vaccine reacts with the test reagent, and the content of each component of the vaccine is determined by a standard curve.
3. The test method according to claim 1, wherein the test method is applied to an adjuvant-adsorbed component vaccine.
4. The detection method according to claim 1, wherein the detection method uses a standard curve method.
5. the detection method according to claim 2, wherein in the first step and the fourth step, the Tween-80 solution is added to the vaccine in a volume of not more than 1% of the total volume.
6. the detection method according to claim 2, wherein in the first step and the fourth step, tween-80 is added to the vaccine at a final concentration of not more than 1%.
7. the detection method according to claim 1,
(1) Taking 495 mu L of pneumococcus conjugate, sequentially adding 5 mu L of 1% Tween-80 solution, 15 mu L of 1mol/L sodium hydroxide solution and 6 mu L of 1mol/L citric acid solution, fully and uniformly mixing in each step, wherein the polysaccharide concentration is 20 mu g/ml, and taking the polysaccharide as a calibration substance working solution;
(2) Diluting the working solution of the calibration standard into a series of concentration gradient solutions with polysaccharide concentration of 5.5 mug/ml, 4.5 mug/ml, 3.5 mug/ml and 2.5 mug/ml by using a diluent as a standard curve;
(3) reacting the standard curve with serum matched with the conjugate, and drawing the standard curve by taking the polysaccharide content as an abscissa and the response value as an ordinate;
(4) and taking 500 mu L of pneumococcal conjugate vaccine, sequentially adding 5 mu L of 1% Tween-80 solution, 15 mu L of 1mol/L sodium hydroxide solution and 6 mu L of 1mol/L citric acid solution, fully and uniformly mixing in each step, then reacting with serum, and determining the content of each component of the vaccine through a standard curve.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111308071A (en) * | 2020-03-13 | 2020-06-19 | 长春生物制品研究所有限责任公司 | Desorption agent for antigen in aluminium salt desorption type vaccine and antigen content detection method |
| 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 |
| CN111840540A (en) * | 2020-08-04 | 2020-10-30 | 华北制药金坦生物技术股份有限公司 | Method for analyzing hepatitis B vaccine |
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| CN111308071A (en) * | 2020-03-13 | 2020-06-19 | 长春生物制品研究所有限责任公司 | Desorption agent for antigen in aluminium salt desorption type vaccine and antigen content detection method |
| CN111308071B (en) * | 2020-03-13 | 2021-06-18 | 长春生物制品研究所有限责任公司 | Desorption agent for antigen in aluminium salt desorption type vaccine and antigen content detection method |
| 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 |
| CN111812313B (en) * | 2020-06-22 | 2021-10-08 | 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 |
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