CN113607939A - Method for measuring adsorption rate of various combined stock solutions of pneumococcal polysaccharide combined vaccine - Google Patents
Method for measuring adsorption rate of various combined stock solutions of pneumococcal polysaccharide combined vaccine Download PDFInfo
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 58
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 58
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 58
- 238000001179 sorption measurement Methods 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 42
- 239000011550 stock solution Substances 0.000 title claims abstract description 35
- 229960005486 vaccine Drugs 0.000 title description 7
- 238000012360 testing method Methods 0.000 claims abstract description 90
- 238000006243 chemical reaction Methods 0.000 claims abstract description 63
- 238000002965 ELISA Methods 0.000 claims abstract description 48
- 239000000243 solution Substances 0.000 claims abstract description 44
- 102000004190 Enzymes Human genes 0.000 claims abstract description 35
- 108090000790 Enzymes Proteins 0.000 claims abstract description 35
- 108010060123 Conjugate Vaccines Proteins 0.000 claims abstract description 22
- 229940031670 conjugate vaccine Drugs 0.000 claims abstract description 22
- 238000000576 coating method Methods 0.000 claims abstract description 20
- 239000011248 coating agent Substances 0.000 claims abstract description 17
- 238000007789 sealing Methods 0.000 claims abstract description 11
- 239000000758 substrate Substances 0.000 claims abstract description 10
- 238000012545 processing Methods 0.000 claims abstract description 5
- 239000000523 sample Substances 0.000 claims description 211
- 239000011534 wash buffer Substances 0.000 claims description 28
- 238000005406 washing Methods 0.000 claims description 20
- 241000283707 Capra Species 0.000 claims description 15
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 12
- 229960005367 tetanus antitoxin Drugs 0.000 claims description 11
- 238000007865 diluting Methods 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000006911 enzymatic reaction Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000002372 labelling Methods 0.000 claims description 5
- 239000012470 diluted sample Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims 1
- 238000011161 development Methods 0.000 abstract description 3
- 239000000427 antigen Substances 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 9
- 102000014914 Carrier Proteins Human genes 0.000 description 8
- 108010078791 Carrier Proteins Proteins 0.000 description 8
- 206010043376 Tetanus Diseases 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 7
- 239000007790 solid phase Substances 0.000 description 6
- 206010035664 Pneumonia Diseases 0.000 description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
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- 230000001900 immune effect Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
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- 238000005303 weighing Methods 0.000 description 3
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 2
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
- G01N2333/3156—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
Abstract
The invention discloses a method for measuring adsorption rates of various types of conjugate stock solutions of pneumococcal polysaccharide conjugate vaccines, which comprises the following steps: processing a sample to be tested to obtain a target sample A; coating and sealing the ELISA plate; respectively adding the diluted test sample and the target sample A into the coated and sealed enzyme label plate to obtain a test sample of the test sample and a test sample of the target sample A; adding specific antiserum into the test sample of the test sample and the test sample of the target sample A to obtain a reaction sample of the test sample and a reaction sample of the target sample A; respectively adding enzyme-labeled antibodies into a test sample reaction sample and a target sample A reaction sample, then adding a substrate solution for color development reaction, and measuring an OD value; and calculating the corresponding polysaccharide content according to the OD values obtained in the test sample reaction sample and the target sample A reaction sample, thereby calculating the adsorption rate.
Description
Technical Field
The invention relates to a method for measuring adsorption rate by using various types of polysaccharide and carrier protein through chemical combination, then adsorbing polysaccharide protein combination on a solid phase carrier through a coating protein antibody, and then reacting serum of various types of specific antibodies with various types of polysaccharide, in particular to a method for measuring adsorption rate of various types of combined stock solution of pneumococcal polysaccharide combined vaccine.
Background
After the antigen is adsorbed by the adjuvant, the antigen is highly aggregated on the surface and in the adjuvant, the process does not change the physical and chemical properties of the antigen, and the antigen can be physically presented to immune cells, so that the immune cells are promoted to be contacted with the antigen fully, at a high level and for a long time, and an immune response is induced, and the process is called as 'storage effect'. However, the adsorption capacity of the adjuvant is closely related to the adsorption rate of the adjuvant to the antigen, and the stronger the adsorption capacity is, the higher the adsorption rate to the antigen is. The adsorption rate of the adjuvant to the antigen is closely related to the immune effect of the vaccine, and the higher the adsorption rate of the antigen and the adjuvant is, the better the immune effect is; conversely, the poorer the immune effect. Therefore, only by ensuring that the adjuvant has strong adsorption capacity, the high adsorption rate of the antigen can be realized, and finally, the vaccine has good immune effect.
The existing method for determining the adsorption rate of the vaccine only measures the total adsorption rate of the pneumococcal polysaccharide conjugate vaccine, but cannot measure the respective adsorption rate of each serotype of various conjugate stock solutions of the pneumococcal polysaccharide conjugate vaccine.
Therefore, a method for measuring the adsorption rate of various types of conjugate stock solutions of pneumococcal polysaccharide conjugate vaccines is needed to solve the above problems
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide a method for measuring adsorption rates of various binding stock solutions of pneumococcal polysaccharide conjugate vaccines, which can measure the adsorption rates of the various binding stock solutions of the pneumococcal polysaccharide conjugate vaccines respectively.
In order to achieve the above object, an embodiment of the present invention provides a method for determining adsorption rates of various binding stock solutions of pneumococcal polysaccharide conjugate vaccines, which is characterized by comprising the following steps:
processing a sample to be tested to obtain a target sample A;
coating and sealing the ELISA plate;
respectively adding the diluted test sample and the target sample A into the coated and sealed enzyme label plate to obtain a test sample of the test sample and a test sample of the target sample A;
adding specific antiserum into the test sample of the test sample and the test sample of the target sample A to obtain a reaction sample of the test sample and a reaction sample of the target sample A;
respectively adding enzyme-labeled antibodies into a test sample reaction sample and a target sample A reaction sample, incubating, then adding a substrate solution for color reaction, and measuring an OD value;
and calculating the corresponding polysaccharide content according to the OD values obtained in the test sample reaction sample and the target sample A reaction sample, and calculating the adsorption rate according to the polysaccharide content of the test sample reaction sample and the target sample A reaction sample.
In one or more embodiments of the present invention, the sample is centrifuged, and the centrifuged supernatant is the target sample a.
In one or more embodiments of the present invention, the conditions for subjecting the test sample to centrifugation include: the relative eccentricity is (5000-6500) Xg, the temperature is 4-6 ℃, and the duration is 5-10 min.
In one or more embodiments of the invention, the process of coating the microplate comprises: adding tetanus antitoxin diluted by the coating solution into the ELISA plate, then coating for 16-18 h at the temperature of 2-8 ℃, and washing the ELISA plate for 1-2 times by using a washing buffer solution.
In one or more embodiments of the present invention, the blocking process for the microplate comprises: adding a sealing solution into the coated ELISA plate, standing for 1-2 h at the temperature of 36.9-37 ℃, and washing the ELISA plate for 3-4 times by using a washing buffer solution.
In one or more embodiments of the invention, the diluted test sample and the target sample a are respectively added into the coated and sealed elisa plate, and incubated at 36.9-37 ℃ for 1-2 h to obtain a test sample of the test sample and a test sample of the target sample a.
In one or more embodiments of the present invention, the dilution factor of the sample and the target sample a is 400 to 600 times.
In one or more embodiments of the invention, after adding specific antiserum to the test sample of the test sample and the test sample of the target sample a, incubating for 1-1.5 h at 36.9-37 ℃, and washing the elisa plate with a washing buffer solution for 3-4 times to obtain a reaction sample of the test sample and a reaction sample of the target sample a.
In one or more embodiments of the invention, the enzyme-labeled antibody is HRP-labeled goat anti-rabbit IgG or AP-labeled goat anti-rabbit IgG.
In one or more embodiments of the invention, the enzyme-labeled antibody is expressed as a 1: diluting by 5000-8000, adding the diluted sample into a reaction sample of a test sample and a reaction sample of a target sample A, incubating for 1-1.5 h at the temperature of 36.9-37 ℃, and washing the ELISA plate for 5-6 times by using a washing buffer solution.
In one or more embodiments of the present invention, the conditions of the color reaction include: standing for 15-20 min at the temperature of 36.9-37 ℃, then respectively adding sulfuric acid solution to terminate the enzyme reaction, and finally reading the OD value by using an enzyme-labeling instrument.
Compared with the prior art, according to the method for determining the adsorption rate of various binding stock solutions of the pneumococcal polysaccharide conjugate vaccine, a test sample and a target sample A are respectively added into an enzyme label plate which is coated and sealed, incubation is carried out to obtain a test sample of the test sample and a test sample of the target sample A, specific antiserum is respectively added into the test sample of the test sample and the test sample of the target sample A to respectively obtain a reaction sample of the test sample and a reaction sample of the target sample A, enzyme-labeled antibodies are respectively added into the reaction sample of the test sample and the reaction sample of the target sample A, substrate solution is added for color reaction after incubation, an OD value is obtained, the polysaccharide content of the reaction sample of the test sample and the reaction sample of the target sample A is finally obtained through calculation, and the adsorption rate of various binding stock solutions is obtained through calculation according to the polysaccharide content.
Detailed Description
The following detailed description of the present invention will be given with reference to examples, but it should be understood that the scope of the present invention is not limited to the specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
The method for measuring the adsorption rate of each type of binding stock solution of the pneumococcal polysaccharide conjugate vaccine comprises the following steps:
and processing the sample to be tested to obtain a target sample A.
Wherein, the treatment process of the sample can be that the sample is centrifuged, and the centrifuged supernatant is the target sample A.
It should be noted that both the test sample and the target sample A contained the pneumococcal polysaccharide and tetanus carrier protein together.
Specifically, the conditions for centrifuging the sample include: the relative eccentricity is (5000-6500) Xg, the temperature is 4-6 ℃, and the duration is 5-10 min.
And (4) coating and sealing the enzyme label plate.
Wherein, the process of coating and processing the ELISA plate comprises the following steps: adding tetanus antitoxin diluted by the coating solution into the ELISA plate, then coating for 16-18 h at the temperature of 2-8 ℃, and washing the ELISA plate for 1-2 times by using a washing buffer solution. I.e. the coating process is completed.
It should be noted that the purpose of the above procedure is to attach tetanus antitoxin to a solid support (microplate).
In the above coating process, a carbonate buffer solution may be used as the coating solution. The amount of washing buffer used was 300. mu.l/well, wherein the wells were wells of an enzyme-labeled plate.
Wherein, the process of sealing the ELISA plate comprises the following steps: adding a sealing solution into the coated ELISA plate, standing at the temperature of 36.9-37 ℃ for 1-2 h, and washing the ELISA plate for 3-4 times by using a washing buffer solution, thus completing the sealing process.
It should be noted that the purpose of the above procedure is to shield other functional groups in tetanus antitoxin, and only allow the test sample and tetanus carrier protein in target sample A to bind to tetanus antitoxin and be immobilized on the microplate.
In the above blocking procedure, PBS buffer containing 5% BSA or 10% calf serum was used as a blocking solution, and the amount of the blocking solution added to each well of the microplate was 200. mu.l.
And respectively adding the diluted test sample and the target sample A into the coated and sealed enzyme label plate, incubating for 1-2 h at the temperature of 36.9-37 ℃, and combining the test sample of the test sample and the test sample of the target sample A with the tetanus antitoxin adsorbed on the solid phase carrier.
Wherein, the dilution times of the sample to be tested and the target sample A can be 400-600 times. The purpose of the above procedure is to bind tetanus carrier protein in the test sample as well as target sample A to tetanus antitoxin and thus immobilize it on the microplate.
And adding corresponding type specific antiserum into the enzyme-labeled plate holes of the test sample and the target sample A test sample to obtain a test sample reaction sample and a target sample A reaction sample.
The method comprises the following specific steps: adding corresponding type specificity antiserum into the test sample of the test article and the test sample of the target sample A, incubating for 1-1.5 h at the temperature of 36.9-37 ℃, and washing the ELISA plate for 3-4 times by using a washing buffer solution to obtain a reaction sample of the test article and a reaction sample of the target sample A.
It should be noted that specific antisera refer to pneumonia antibodies of each serotype. The purpose of the above process is that the pneumonia polysaccharide in the sample and the target sample A is respectively combined with pneumonia antibody of each serotype, thereby being fixed in the ELISA plate. In order to separate the various types of conjugate stock solutions of the pneumococcal polysaccharide conjugate vaccine, 20 different specific antisera, namely 20 antibodies matched with 20 different serotypes of pneumococcal polysaccharide conjugate vaccines, are prepared in the ELISA plate.
Adding enzyme-labeled antibodies into the reaction sample of the sample to be tested and the reaction sample of the target sample A respectively, incubating, adding substrate solution for color reaction, and measuring the OD value.
Wherein, the specific processes of adding the enzyme-labeled antibody and incubating are as follows: the enzyme-labeled antibody is prepared according to the following steps of 1: diluting by 5000-8000, adding the diluted sample into a reaction sample of a test sample and a reaction sample of a target sample A, incubating for 1-1.5 h at the temperature of 36.9-37 ℃, and washing the ELISA plate for 5-6 times by using a washing buffer solution.
Wherein the enzyme-labeled antibody is HRP-labeled goat anti-rabbit IgG or AP-labeled goat anti-rabbit IgG.
Wherein, the condition of the color reaction comprises: standing for 15-20 min at the temperature of 36.9-37 ℃, then respectively adding sulfuric acid solution to terminate the enzyme reaction, and finally reading the OD value by using an enzyme-labeling instrument.
In the above process, the enzyme-labeled antibody is combined with the antibodies in the sample reaction sample and the target sample a reaction sample to form an antigen-antibody complex, the complex is subjected to a color development reaction with a substrate solution to be added subsequently, and the OD value is measured.
If HRP-labeled goat anti-rabbit IgG is used as an enzyme-labeled antibody, OPD can be used as a substrate solution, and the OD value is read at 490nm by a corresponding enzyme-labeling instrument; the substrate solution can also use TMB, and the OD value is read at 450nm by a corresponding microplate reader.
If AP labeled goat anti-rabbit IgG is used as an enzyme labeled antibody, pNPP can be used as a substrate solution, and the OD value is read at 405nm by a corresponding enzyme labeling instrument.
And calculating the corresponding polysaccharide content according to the OD values obtained in the test sample reaction sample and the target sample A reaction sample, and calculating the adsorption rate according to the polysaccharide content of the test sample reaction sample and the target sample A reaction sample.
In order to calculate the corresponding polysaccharide content from the OD values obtained in the sample reaction sample and the target sample A reaction sample, the binding stock solutions having known polysaccharide concentrations were diluted to 1ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, and 20ng/mL, respectively. And measuring the five groups of combined stock solutions according to the steps to obtain corresponding OD values, then establishing a regression equation, and bringing the OD values in the reaction sample of the test sample and the reaction sample of the target sample A into the equation to obtain the corresponding polysaccharide concentration. And then the adsorption rate of various types of combined stock solutions of the pneumococcal polysaccharide combined vaccine is obtained by substituting the adsorption rate into the following disclosure.
Adsorption rate (%):
wherein, note: h: adsorption rate (%); YS: a target sample A; y: test samples were tested before centrifugation.
In summary, the principle of the method for determining the adsorption rate of various binding stock solutions of the pneumococcal polysaccharide conjugate vaccine of the invention is as follows: the pneumococcal conjugate stock solution is prepared by mixing various pneumococcal derivatives and tetanus carrier protein, and the method mainly utilizes a reverse indirect method to connect a second antibody aiming at the tetanus carrier protein to a solid phase carrier for binding all tetanus carrier protein in the conjugate stock solution, wherein the pneumonia polysaccharide is combined with the tetanus carrier protein, then a specific antibody is added to be combined with the pneumonia polysaccharide in a sample to form a solid phase antigen-detected antibody compound, an enzyme-labeled secondary antibody is combined with an antibody in the solid phase immune compound to form the solid phase antigen-detected antibody-enzyme-labeled secondary antibody compound, the color development degree after adding a substrate is measured, and the content of the antibody to be detected is determined.
In the case of the example 1, the following examples are given,
weighing 5ml of test sample, diluting by 400 times, dividing into two parts, centrifuging one part of the test sample for 5min under the conditions of relative eccentricity of 5000 Xg and temperature of 4 ℃, and taking supernatant after centrifugation treatment to obtain a target sample A. The conjugate stock solutions with known polysaccharide concentrations were diluted to 1ng/mL, 5ng/mL, 10ng/mL, 15ng/mL and 20ng/mL, respectively, and scored as five standards.
And adding tetanus antitoxin diluted by carbonate buffer solution into the ELISA plate, adding 100 mu l/hole and 160 holes in total, then coating for 16h at 2 ℃, and then washing the ELISA plate for 1 time by using washing buffer solution (300 mu l/hole). The coated plate was added to PBS buffer containing 5% BSA at 100. mu.l/well and allowed to stand at 36.9 ℃ for 1 hour. The microplate was washed 3 times with washing buffer (300. mu.l/well).
Each group of 8 wells is divided into 20 groups, and each well in each group of 8 wells is added with 100 μ l of a test sample, a target sample A, five standard samples and one of diluents (serving as a control group), and then the ELISA plate is incubated for 1h at 36.9 ℃, and then washed 3 times with a washing buffer (300 μ l/well).
And (3) respectively adding the diluted 20 types of serum into 20 groups of enzyme-labeled plate holes, wherein each group of enzyme-labeled plate holes is added with only one type of serum, incubating for 1h at the temperature of 36.9 ℃, and washing the enzyme-labeled plate for 3 times by using a washing buffer solution (300 mu l/hole).
Diluting the HRP-labeled goat anti-rabbit IgG according to the proportion of 1:5000, adding the diluted HRP-labeled goat anti-rabbit IgG into an ELISA plate, incubating for 1h at 36.9 ℃, and washing the ELISA plate for 5 times by using a washing buffer solution (300 mu l/hole). Then, 100. mu.l of OPD was added to each well of the plate, and after standing at 36.9 ℃ for 15min, the enzyme reaction was stopped with 50. mu.l/well of 2mol/L sulfuric acid solution, and the OD value was read at 490nm in a plate reader.
And finally, calculating a regression equation corresponding to the OD value of each group and the polysaccharide concentration according to the OD values of the five standard samples in each group of the ELISA plate holes, substituting the OD values corresponding to the test sample and the target sample A into the regression equation to obtain the corresponding polysaccharide concentration, and substituting the polysaccharide concentration into an adsorption rate formula to obtain the adsorption rate of the serotype binding stock solution corresponding to each group of the ELISA plate holes.
In the case of the example 2, the following examples are given,
weighing 5ml of test sample, diluting by 500 times, dividing into two parts, centrifuging one part of the test sample for 85min under the conditions of the relative eccentricity of 6000 Xg and the temperature of 5 ℃, and taking supernatant after centrifugation treatment to obtain the target sample A. The conjugate stock solution with known polysaccharide concentration was further weighed and diluted to 1ng/mL, 5ng/mL, 10ng/mL, 15ng/mL and 20ng/mL respectively, and recorded as five standards.
And adding tetanus antitoxin diluted by carbonate buffer solution into the ELISA plate, adding 100 mu l/hole and 160 holes in total, then coating for 17h at 5 ℃, and then washing the ELISA plate for 2 times by using washing buffer solution (300 mu l/hole). The coated ELISA plate was added to PBS buffer containing 10% calf serum at 100. mu.l/well and allowed to stand at 36.9 ℃ for 1.5 hours. The microplate was washed 3 times with washing buffer (300. mu.l/well).
Each group of 8 wells is divided into 20 groups, and each well in each group of 8 wells is added with 100 μ l of a test sample, a target sample A, five standard samples and one of diluents (serving as a control group), and then the ELISA plate is incubated for 1.5h at 37 ℃ and washed 3 times with a washing buffer (300 μ l/well).
And (3) respectively adding the diluted 20 types of serum into 20 groups of enzyme-labeled plate holes, wherein each group of enzyme-labeled plate holes is added with only one type of serum, incubating for 1.2h at 37 ℃, and washing the enzyme-labeled plate for 3 times by using a washing buffer solution (300 mu l/hole).
Diluting the HRP-labeled goat anti-rabbit IgG according to the proportion of 1:6500, adding the diluted HRP-labeled goat anti-rabbit IgG into an ELISA plate, incubating for 1.2h at the temperature of 36.9 ℃, and washing the ELISA plate for 5 times by using a washing buffer solution (300 mu l/hole). Then, 100. mu.l of TMB was added to each well of the microplate, and after standing at 37 ℃ for 18min, the enzyme reaction was terminated with 50. mu.l/well of 2mol/L sulfuric acid solution, and the OD value was read at 450nm in a microplate reader.
And finally, calculating a regression equation corresponding to the OD value of each group and the polysaccharide concentration according to the OD values of the five standard samples in each group of the ELISA plate holes, substituting the OD values corresponding to the test sample and the target sample A into the regression equation to obtain the corresponding polysaccharide concentration, and substituting the polysaccharide concentration into an adsorption rate formula to obtain the adsorption rate of the serotype binding stock solution corresponding to each group of the ELISA plate holes.
In the case of the example 3, the following examples are given,
weighing 5ml of test sample, diluting by 600 times, dividing into two parts, centrifuging one part of the test sample for 10min under the conditions of the relative eccentricity of 6500 Xg and the temperature of 6 ℃, and taking supernatant after centrifugation, namely the target sample A. The conjugate stock solution with known polysaccharide concentration was further weighed and diluted to 1ng/mL, 5ng/mL, 10ng/mL, 15ng/mL and 20ng/mL respectively, and recorded as five standards.
And adding tetanus antitoxin diluted by carbonate buffer solution into the ELISA plate, adding 100 mu l/hole and 160 holes in total, then coating for 18h at 8 ℃, and then washing the ELISA plate for 1 time by using washing buffer solution (300 mu l/hole). The coated plate was added to PBS buffer containing 5% BSA at 100. mu.l/well and allowed to stand at 37 ℃ for 2 hours. The microplate was labeled 4 times with washing buffer (300. mu.l/well).
The 8 wells are divided into 20 groups, each group of 8 wells is added with 100 μ l of the test sample, the target sample A, five standard samples and one of the diluents (as a control group) respectively, then the incubation is carried out for 2h at 37 ℃, and the ELISA plate is washed 4 times by using the washing buffer (300 μ l/well).
And (3) respectively adding the diluted 20 types of serum into 20 groups of enzyme-labeled plate holes, wherein each group of enzyme-labeled plate holes is added with only one type of serum, incubating for 1.5h at 37 ℃, and washing the enzyme-labeled plate for 4 times by using a washing buffer solution (300 mu l/hole).
Diluting AP-labeled goat anti-rabbit IgG according to the ratio of 1:8000, adding the diluted AP-labeled goat anti-rabbit IgG into an ELISA plate, incubating the diluted AP-labeled goat anti-rabbit IgG for 1.5h at the temperature of 37 ℃, and washing the ELISA plate for 6 times by using a washing buffer solution (300 mu l/hole). Then, 100. mu.l of pNPP was added to each well of the plate, and after standing at 37 ℃ for 20min, the enzyme reaction was terminated with 50. mu.l/well of 2mol/L sulfuric acid solution, and the OD value was read at 405nm in a plate reader.
And finally, calculating a regression equation corresponding to the OD value of each group and the polysaccharide concentration according to the OD values of the five standard samples in each group of the ELISA plate holes, substituting the OD values corresponding to the test sample and the target sample A into the regression equation to obtain the corresponding polysaccharide concentration, and substituting the polysaccharide concentration into an adsorption rate formula to obtain the adsorption rate of the serotype binding stock solution corresponding to each group of the ELISA plate holes.
The data obtained for the three examples are shown in the following table:
the adsorption rates of various binding stocks in pneumococcal polysaccharide conjugate vaccines can be clearly known from the data in the table. In example 1, the polysaccharide concentration of the target sample a in the 33F type binding stock solution data is negative, which results in the calculated adsorption rate value exceeding 100%, and an error occurs, but the error is within an allowable error range and can be ignored, and the measurement effect of the method is not influenced.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (10)
1. A method for measuring adsorption rate of various binding stock solutions of pneumococcal polysaccharide conjugate vaccines is characterized by comprising the following steps:
processing a sample to be tested to obtain a target sample A;
coating and sealing the ELISA plate;
respectively adding the diluted test sample and the target sample A into the coated and sealed enzyme label plate to obtain a test sample of the test sample and a test sample of the target sample A;
adding specific antiserum into the test sample of the test sample and the test sample of the target sample A to obtain a reaction sample of the test sample and a reaction sample of the target sample A;
respectively adding enzyme-labeled antibodies into a test sample reaction sample and a target sample A reaction sample, incubating, then adding a substrate solution for color reaction, and measuring an OD value;
and calculating the corresponding polysaccharide content according to the OD values obtained in the test sample reaction sample and the target sample A reaction sample, and calculating the adsorption rate according to the polysaccharide content of the test sample reaction sample and the target sample A reaction sample.
2. The method for measuring the adsorption rate of each binding stock solution of pneumococcal polysaccharide conjugate vaccine according to claim 1, wherein the sample is centrifuged, and the centrifuged supernatant is the target sample A.
3. The method of claim 2, wherein the conditions for subjecting the sample to centrifugation comprise: the relative eccentricity is (5000-6500) Xg, the temperature is 4-6 ℃, and the duration is 5-10 min.
4. The method for measuring the adsorption rate of each type of conjugate stock solution of pneumococcal polysaccharide conjugate vaccine according to claim 1, wherein the coating process for the ELISA plate comprises: adding tetanus antitoxin diluted by coating liquid into an ELISA plate, then coating for 16-18 h under the condition of 2-8 ℃, washing the ELISA plate for 1-2 times by using washing buffer solution, and carrying out sealing treatment on the ELISA plate, wherein the sealing treatment process comprises the following steps: adding a sealing solution into the coated ELISA plate, standing for 1-2 h at the temperature of 36.9-37 ℃, and washing the ELISA plate for 3-4 times by using a washing buffer solution.
5. The method for measuring the adsorption rate of each type of binding stock solution of pneumococcal polysaccharide conjugate vaccine according to claim 1, wherein the diluted test sample and the target sample A are respectively added into the coated and sealed enzyme label plate, and incubated at 36.9-37 ℃ for 1-2 h to obtain the test sample of the test sample and the test sample of the target sample A.
6. The method for measuring the adsorption rate of each binding stock solution of pneumococcal polysaccharide conjugate vaccine according to claim 5, wherein the dilution factor of the sample and the target sample A is 400-600 times.
7. The method for measuring the adsorption rate of each binding stock solution of the pneumococcal polysaccharide conjugate vaccine according to claim 1, wherein the test sample of the sample to be tested and the test sample of the target sample A are respectively added with specific antiserum, incubated for 1-1.5 h at 36.9-37 ℃, and the ELISA plate is washed for 3-4 times by using a washing buffer solution to obtain the reaction sample of the sample to be tested and the reaction sample of the target sample A.
8. The method for measuring the adsorption rate of each type of binding stock solution of pneumococcal polysaccharide conjugate vaccine of claim 1, wherein the enzyme-labeled antibody is HRP-labeled goat anti-rabbit IgG or AP-labeled goat anti-rabbit IgG.
9. The method for measuring the adsorption rate of each type of conjugate stock solution of pneumococcal polysaccharide conjugate vaccine according to claim 8, wherein the enzyme-labeled antibody is labeled according to a ratio of 1: diluting by 5000-8000, adding the diluted sample into a reaction sample of a test sample and a reaction sample of a target sample A, incubating for 1-1.5 h at the temperature of 36.9-37 ℃, and washing the ELISA plate for 5-6 times by using a washing buffer solution.
10. The method for measuring the adsorption rate of each type of conjugate stock solution of pneumococcal polysaccharide conjugate vaccine according to claim 1, wherein the condition of the color reaction comprises: standing for 15-20 min at the temperature of 36.9-37 ℃, then respectively adding sulfuric acid solution to terminate the enzyme reaction, and finally reading the OD value by using an enzyme-labeling instrument.
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| US20030099672A1 (en) * | 2000-03-17 | 2003-05-29 | Dominique Schultz | Pneumococcus polysaccharide conjugates for use as vaccine against tetanus an diphtheria |
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