CN106771016B - Rapid qualitative and quantitative detection method for oil adjuvant vaccine - Google Patents
Rapid qualitative and quantitative detection method for oil adjuvant vaccine Download PDFInfo
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- CN106771016B CN106771016B CN201611122911.7A CN201611122911A CN106771016B CN 106771016 B CN106771016 B CN 106771016B CN 201611122911 A CN201611122911 A CN 201611122911A CN 106771016 B CN106771016 B CN 106771016B
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- 229960005486 vaccine Drugs 0.000 title claims abstract description 48
- 238000001514 detection method Methods 0.000 title claims abstract description 35
- 239000002671 adjuvant Substances 0.000 title claims abstract description 26
- 239000000427 antigen Substances 0.000 claims abstract description 57
- 102000036639 antigens Human genes 0.000 claims abstract description 57
- 108091007433 antigens Proteins 0.000 claims abstract description 57
- 239000000523 sample Substances 0.000 claims abstract description 54
- 238000000034 method Methods 0.000 claims abstract description 32
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 238000000746 purification Methods 0.000 claims abstract description 11
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 5
- 239000012521 purified sample Substances 0.000 claims abstract description 5
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 claims description 5
- 238000001962 electrophoresis Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 3
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 2
- 239000004472 Lysine Substances 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 238000010186 staining Methods 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 20
- 239000012535 impurity Substances 0.000 abstract description 7
- 238000011084 recovery Methods 0.000 abstract description 5
- 239000004094 surface-active agent Substances 0.000 abstract description 5
- 230000010355 oscillation Effects 0.000 abstract description 2
- HRHKULZDDYWVBE-UHFFFAOYSA-N indium;oxozinc;tin Chemical compound [In].[Sn].[Zn]=O HRHKULZDDYWVBE-UHFFFAOYSA-N 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 21
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 description 9
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 7
- 230000010354 integration Effects 0.000 description 7
- 239000008346 aqueous phase Substances 0.000 description 6
- 229940023041 peptide vaccine Drugs 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 2
- 230000000091 immunopotentiator Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
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- 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
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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Abstract
The invention provides a rapid qualitative and quantitative detection method of an oil adjuvant vaccine, which is characterized by comprising the following steps: demulsifying the oil adjuvant vaccine, carrying out ZIPTIP purification on the obtained water-phase antigen sample, and carrying out qualitative and quantitative detection on the purified sample; the demulsification method comprises the following steps: and mixing the oil adjuvant vaccine with n-butanol, adding a competitor, uniformly mixing by oscillation, and centrifuging to obtain a water-phase antigen sample. The method has the advantages that the antigen binding sites are competed with the surfactant by adding the competition agent, so that the antigen in the oil adjuvant vaccine is released into the water phase, and the antigen recovery rate in the water phase is greatly improved; and purifying the demulsified sample by ZIPTIP (zinc-indium-tin oxide), so that impurities in the sample are effectively removed, the purity of the sample is greatly improved, and the sample is qualitatively and quantitatively analyzed, so that the accuracy and reliability of a detection result are improved.
Description
Technical Field
The invention relates to the technical field of foot-and-mouth disease vaccine detection, in particular to a rapid qualitative and quantitative detection method of an oil adjuvant vaccine.
Background
The existing vaccine quality standard stipulates that the efficacy test must be carried out by the animal, because the state carries out 100% of intensified immunity policy, the susceptible test animal is difficult to select, the animal toxicity attacking has high requirements on experimental facilities (BS L3 grade laboratory), the time consumption is long (more than one month), and the capital cost is high.
The current state inspection of vaccines is gradually transiting to the detection of antigens in vaccines, and the current common method is to detect the antigens after the demulsification of the vaccines, transfer the antigens to a water phase by performing the demulsification treatment on the vaccines, and then perform subsequent detection and analysis on the antigens. As is known, the vaccine is prepared by emulsifying an antigen and an adjuvant according to a certain proportion and a specific program, but the adjuvant brings great obstacles for demulsification detection of the vaccine.
Because the oil adjuvant used in the emulsification process of the vaccine contains complex components and contains substances such as a surfactant, an immunopotentiator and the like, the water phase after demulsification often contains the impurities and the demulsifier, a method for removing the impurities and the demulsifier is not available in the industry, the impurities, the demulsifier and the like can greatly influence the subsequent detection process, so that signal masking or interference is caused, the strength of an antigen signal is reduced, even the antigen in the antigen can not be effectively detected, the repeatability of the detection method is poor due to the random distribution of the impurities in the antigen, and the maintenance cost of an instrument is increased. In the vaccine industry, how to demulsify an oil adjuvant vaccine and detect and identify components in the vaccine is a technical difficulty recognized in the industry, and because the oil adjuvant vaccine components are complex and contain a large amount of substances such as surfactants, immunopotentiators and the like, interference can be caused to all detection methods, even the detection cannot be carried out, and the real state of the antigen in the vaccine cannot be reflected.
Although the existing demulsification methods in the industry have poor efficiency and effect, the traditional demulsification methods have various problems after demulsification, such as incomplete demulsification, unclear separation of a water phase and an oil phase, no antigen detected in the water phase and the like. How to find an efficient demulsification method becomes an urgent problem to be solved in the industry.
The ZIPTIP method is a simple method for desalting before mass spectrometry, and is used for removing salt substances in analysis and detection. There is no report in the prior art of using this method for antigen purification.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a rapid qualitative and quantitative detection method of an oil adjuvant vaccine.
The purpose of the invention is realized by the following technical scheme:
the invention provides a rapid qualitative and quantitative detection method of an oil adjuvant vaccine, which comprises the following steps:
demulsifying the oil adjuvant vaccine, carrying out ZIPTIP purification on the obtained water-phase antigen sample, and carrying out qualitative and quantitative detection on the purified sample;
the demulsification method comprises the following steps: and mixing the oil adjuvant vaccine with n-butanol, adding a competitor, uniformly mixing by oscillation, and centrifuging to obtain a water-phase antigen sample.
Preferably, the competitor comprises at least one of an amino acid and a derivative thereof.
Preferably, the competitor is one of lysine, arginine, phenylalanine, histidine and proline.
Preferably, the amount of the competitor added is: 1-40mg of competitor, more preferably 1-20mg of competitor, is added per 1ml of oil-adjuvanted vaccine. The concentration of the competitor is too high, so that the competitor is saturated and separated out, and the ultrafiltration process is influenced; too low a concentration may result in poor competition and insufficient release of antigen for detection.
Preferably, the volume ratio of the oil-adjuvanted vaccine to n-butanol is 9: 1-5: 5.
more preferably, the volume ratio of the oil-adjuvanted vaccine to n-butanol is 1: 1. The volume of the oil adjuvant vaccine is the same as that of the n-butyl alcohol, so that complete demulsification of the vaccine can be better ensured.
Preferably, the specific steps of purifying ZIPTIP are as follows:
A1. using 50% ACN to suction activate zipIP;
A2. sucking the sample for several times;
A3. blotting and washing ZIPTIP with 0.05% TFA water;
A4. eluting the sample with ACN containing 0.05% TFA.
Preferably, the purified sample is lyophilized or concentrated.
Preferably, the antigen sample is quantitatively and qualitatively detected by a method of staining after electrophoresis.
By adopting the demulsification method, the antigen recovery rate is high, and the obtained antigen sample can be directly used for quantitative and qualitative detection of the antigen without further purification.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention releases the antigen in the oil adjuvant vaccine into the water phase by adding the competition agent and the surfactant to compete for the antigen binding site, and greatly improves the antigen recovery rate in the water phase compared with the method without adding the competition agent.
2) The ZIPTIP purification method is adopted, so that impurities in an antigen sample can be effectively removed, the impurities such as a surfactant and the like in a complex sample can be removed through specific adsorption of the antigen, and the sample obtained after elution can be directly used for analysis and detection of a high-precision instrument.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a graph showing the detection of the antigen concentration HP L C in the aqueous phase after demulsification by the method of comparative example 1.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1
The embodiment provides a rapid qualitative and quantitative detection method of an oil adjuvant vaccine, which comprises the following steps:
1) sample demulsification is carried out according to the following method:
mixing 10ml of vaccine to be detected (commercial pig foot-and-mouth disease synthetic peptide vaccine with the concentration of 75ug/ml) with n-butanol according to the volume ratio of 1:1, adding 50mg of histidine, shaking and mixing uniformly, centrifuging at 4 ℃ for 15 minutes at 3000r/min, and carefully extracting the lower water phase by using a 10ml syringe after centrifuging to obtain the water phase antigen sample.
2) ZIPTIP purification of samples
2.1 freeze-drying or concentrating the aqueous phase antigen sample prepared in the step 1), and purifying by using ZIPTIP, wherein the steps are as follows:
1. using 50% ACN to suction activate zipIP;
2. sucking the sample for several times;
3. blotting and washing ZIPTIP with 0.05% TFA water;
4. samples were eluted using ACN with 0.05% TFA.
3) Qualitative and quantitative detection
The volume of the sample purified by the step 2) is 1/10 (namely 0.5ml) of the water phase after demulsification, namely the concentration is increased by 10 times, the sample is dyed after electrophoresis, the band is analyzed, compared with the theoretical molecular weight, the antigen band appears in the region with the theoretical molecular weight, the primary qualitative determination can be carried out on the antigen band, the protein amount of the sample is about 10-15ug according to the gray value of the band, the sample loading amount of the sample is 10ul, the concentration of the sample is 1-1.5mg/ml, the concentration multiple (divided by 10) and the original concentration of the volume change (divided by 2) after demulsification are 50-75ug/ml according to the result of the concentration 69.3ug/ml measured by liquid chromatography.
The commercial pig foot-and-mouth disease synthetic peptide vaccine adopted in the embodiment is compared with a theoretical antigen concentration standard, the peak emergence position of the HP L C detection map sample is integrated, the integration information is shown in table 1, the antigen content in the sample is represented in the form of an integration peak area, and the integration peak area is 2738690. the peak emergence position of the HP L C detection map sample of the antigen sample concentration in the water phase after the emulsion breaking of Butanol + His is integrated, the integration information is shown in table 2, the antigen content in the sample is represented in the form of an integration peak area, and the integration peak area is 2530549. the results in tables 1 and 2 are compared, and the antigen recovery rate is 92.4%, namely the emulsion breaking efficiency is 92.4% after the integration information is compared.
TABLE 1
TABLE 2
Example 2
The embodiment provides a rapid qualitative and quantitative detection method of an oil adjuvant vaccine, which comprises the following steps:
1) sample demulsification is carried out according to the following method:
mixing 10ml of vaccine to be detected (commercial pig foot-and-mouth disease synthetic peptide vaccine with the concentration of 75ug/ml) with n-butanol according to the volume ratio of 1:1, adding 10mg of phenylalanine, shaking and mixing uniformly, centrifuging at 4 ℃ for 15 minutes at 3000r/min, and carefully extracting the lower water phase by using a 10ml syringe after centrifuging to obtain the water phase antigen sample.
The method of the embodiment is adopted to carry out demulsification on the foot-and-mouth disease vaccine, and the demulsification efficiency is 87.6%.
2) ZIPTIP purification of samples
2.1 freeze-drying or concentrating the aqueous phase antigen sample prepared in the step 1), and purifying by using ZIPTIP, wherein the steps are as follows:
1. using 50% ACN to suction activate zipIP;
2. sucking the sample for several times;
3. blotting and washing ZIPTIP with 0.05% TFA water;
4. samples were eluted using ACN with 0.05% TFA.
3) Qualitative and quantitative detection
The volume of the sample purified by the step 2) is 1/10 (namely 0.5ml) of the water phase after demulsification, namely the concentration is increased by 10 times, the sample is dyed after electrophoresis, the band is analyzed, compared with the theoretical molecular weight, the antigen band appears in the region with the theoretical molecular weight, the primary qualitative determination can be carried out on the antigen band, the protein amount of the sample is about 10-15ug according to the gray value of the band, the sample loading amount of the sample is 10ul, the concentration of the sample is 1-1.5mg/ml, the concentration multiple (divided by 10) and the original concentration of the volume change (divided by 2) after demulsification are 50-75ug/ml according to the result of the concentration of 65.7ug/ml measured by liquid chromatography.
Example 3
The embodiment provides a rapid qualitative and quantitative detection method of an oil adjuvant vaccine, which comprises the following steps:
1) sample demulsification is carried out according to the following method:
mixing 10ml of vaccine to be detected (commercial pig foot-and-mouth disease synthetic peptide vaccine with the concentration of 75ug/ml) with n-butanol according to the volume ratio of 1:1, adding 200mg of proline into each tube, shaking and mixing uniformly, centrifuging at 4 ℃ for 15 minutes at 3000r/min, and carefully extracting the lower aqueous phase by using a 10ml syringe after centrifugation to obtain an aqueous phase antigen sample.
The method of the embodiment is adopted to carry out demulsification on the foot-and-mouth disease vaccine, and the demulsification efficiency is 94.8%.
2) ZIPTIP purification of samples
2.1 freeze-drying or concentrating the aqueous phase antigen sample prepared in the step 1), and purifying by using ZIPTIP, wherein the steps are as follows:
1. using 50% ACN to suction activate zipIP;
2. sucking the sample for several times;
3. blotting and washing ZIPTIP with 0.05% TFA water;
4. samples were eluted using ACN with 0.05% TFA.
3) Qualitative and quantitative detection
The volume of the sample purified by the step 2) is 1/10 (namely 0.5ml) of the water phase after demulsification, namely the concentration is increased by 10 times, the sample is dyed after electrophoresis, the strip is analyzed, compared with the theoretical molecular weight, the antigen strip appears in the region with the theoretical molecular weight, the preliminary characterization can be carried out, the protein amount is about 10-15ug according to the grey value of the strip, the sample loading amount is 10ul, the concentration of the sample is 1-1.5mg/ml, the concentration multiple (divided by 10) and the original concentration of the volume change (divided by 2) after demulsification are 50-75ug/ml according to the result of the concentration 71.7ug/ml measured by liquid chromatography.
Comparative example 1
This comparative example provides a demulsification process for a foot-and-mouth disease vaccine, substantially the same as that of example 1, except that: no competitor was added in this comparative example.
The foot-and-mouth disease vaccine is demulsified by adopting the method of the comparative example, the detection map of the antigen concentration HP L C in the water phase after the demulsification of the n-butanol is shown in figure 1, and it can be seen from the figure that no antigen is detected within the theoretical retention time (23-28min) of the antigen, which indicates that the amount of the antigen contained in the sample is extremely low, namely the recovery rate of the antigen in the comparative example is basically zero, the demulsification efficiency is basically zero, and the demulsification efficiency is 0%.
The results of the comparative example and the example show that the antigen of the synthetic peptide vaccine cannot be distributed in the water phase by using the traditional n-butanol demulsification method, the antigen amount detected in the water phase is extremely low and cannot meet the detection requirement, and the antigen amount in the water phase after the demulsification is carried out after the competitor is added reaches more than 85 percent and is enough to meet the subsequent purification detection requirement.
The invention has many applications, and the above description is only a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made without departing from the principles of the invention and these modifications are to be considered within the scope of the invention.
Claims (4)
1. A method for quickly, qualitatively and quantitatively detecting an oil adjuvant vaccine is characterized by comprising the following steps:
demulsifying the oil adjuvant vaccine, carrying out ZIPTIP purification on the obtained water-phase antigen sample, and carrying out qualitative and quantitative detection on the purified sample;
the demulsification method comprises the following steps: mixing the oil adjuvant vaccine with n-butanol, adding a competitor, shaking, mixing uniformly, and centrifuging to obtain a water-phase antigen sample;
the competition agent is one of lysine, arginine, phenylalanine, histidine and proline;
the addition amount of the competition agent is as follows: 1-40mg of competitor was added per 1ml of oil-adjuvanted vaccine.
2. The method for rapidly, qualitatively and quantitatively detecting the oil-adjuvant vaccine according to claim 1, wherein the ZIPTIP purification comprises the following specific steps:
A1. using 50% ACN to suction activate zipIP;
A2. sucking the sample for several times;
A3. blotting and washing ZIPTIP with 0.05% TFA water;
A4. eluting the sample with ACN containing 0.05% TFA.
3. The method for rapid qualitative and quantitative detection of oil-adjuvanted vaccines according to claim 1 wherein the purified samples are lyophilized or concentrated.
4. The method for rapidly, qualitatively and quantitatively detecting an oil-adjuvant vaccine according to claim 1, wherein the antigen sample is quantitatively and qualitatively detected by a method of staining after electrophoresis.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2365020A (en) * | 2000-07-21 | 2002-02-13 | Associated Octel Company Ltd T | Demulsifiying compositions |
| CN104634891A (en) * | 2015-01-19 | 2015-05-20 | 中国科学院过程工程研究所 | Method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S |
| CN105467138A (en) * | 2015-12-04 | 2016-04-06 | 中国农业科学院兰州兽医研究所 | Method for determining components and estimating anti-gen content of foot-and-mouth disease vaccine |
| CN106769358A (en) * | 2016-12-08 | 2017-05-31 | 申联生物医药（上海）股份有限公司 | The purification process of water phase after a kind of oil-adjuvant vaccine demulsification |
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|---|---|---|---|---|
| GB2365020A (en) * | 2000-07-21 | 2002-02-13 | Associated Octel Company Ltd T | Demulsifiying compositions |
| CN104634891A (en) * | 2015-01-19 | 2015-05-20 | 中国科学院过程工程研究所 | Method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S |
| CN105467138A (en) * | 2015-12-04 | 2016-04-06 | 中国农业科学院兰州兽医研究所 | Method for determining components and estimating anti-gen content of foot-and-mouth disease vaccine |
| CN106769358A (en) * | 2016-12-08 | 2017-05-31 | 申联生物医药（上海）股份有限公司 | The purification process of water phase after a kind of oil-adjuvant vaccine demulsification |
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| Title |
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| 黄芪甲苷单克隆抗体的制备及鉴定;于生兰 等;《天然产物研究与开发》;20131130;第25卷;第1568-1571页 * |
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