CN113680105A - Oil adjuvant vaccine demulsifier and application thereof - Google Patents
Oil adjuvant vaccine demulsifier and application thereof Download PDFInfo
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- CN113680105A CN113680105A CN202111025058.8A CN202111025058A CN113680105A CN 113680105 A CN113680105 A CN 113680105A CN 202111025058 A CN202111025058 A CN 202111025058A CN 113680105 A CN113680105 A CN 113680105A
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- demulsifier
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- oil adjuvant
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- 229960005486 vaccine Drugs 0.000 title claims abstract description 38
- 239000002671 adjuvant Substances 0.000 title claims abstract description 22
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000427 antigen Substances 0.000 claims abstract description 14
- 102000036639 antigens Human genes 0.000 claims abstract description 14
- 108091007433 antigens Proteins 0.000 claims abstract description 14
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 12
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 12
- 239000000839 emulsion Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000011084 recovery Methods 0.000 claims abstract description 6
- 230000002051 biphasic effect Effects 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 239000006228 supernatant Substances 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 26
- 239000008346 aqueous phase Substances 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 230000000694 effects Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 241000714201 Feline calicivirus Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008476 aike Substances 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 239000010775 animal oil Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D17/00—Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion
- B01D17/02—Separation of non-miscible liquids
- B01D17/04—Breaking emulsions
-
- 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
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- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Thermal Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a biphase oil adjuvant vaccine demulsifier, which comprises chloroform and ammonium sulfate, wherein the volume ratio of the chloroform to the vaccine emulsion is 2: 1-2, wherein the mass volume ratio of the ammonium sulfate to the vaccine emulsion is 1: 5-20. The method can effectively and quickly separate the water phase and the oil phase in the dual-phase oil adjuvant vaccine, has short demulsification time, less used reagents, simple operation and low requirement on the operation environment, can be operated at room temperature, separates the water phase into supernatant, has obvious water phase, is convenient for extracting the water phase, and can obviously improve the recovery of the antigen.
Description
Technical Field
The invention relates to a biphase oil adjuvant vaccine demulsifier, a preparation method and application thereof, belonging to the technical field of biology.
Background
The quality of the oil adjuvant vaccine which is efficient and rapid is always the focus of attention of people, the efficacy test specified in the existing vaccine quality standard must be carried out by adopting the animal, the animal for susceptible test is difficult to select due to the fact that the state carries out 100% of intensified immunity policy, and the animal virus attack has high requirements on experimental facilities (BSL 3-grade laboratory), long time consumption (more than one month) and large capital cost. If susceptible animals are selected by adopting a serum neutralization test, the non-susceptible animals with cellular immunity are difficult to technically exclude, and the problem of irregular test data often occurs in the test practice, so that the test accuracy is influenced. Therefore, the national vaccine test is slowly converted into the detection of the antigen in the vaccine to replace the existing animal test. The existing demulsification method and demulsifier are not suitable for all oil adjuvant vaccines, and the traditional demulsifier is used for demulsifying the oil adjuvant vaccines, so that the vaccines can be demulsified, but the antigen content in the water phase is extremely low. The demulsification efficiency and the demulsification effect of different demulsifiers are different in level. The invention has short demulsification time, can finish demulsification after the demulsifier is added, can extract the water phase after centrifugation, and can obviously improve the recovery of the antigen.
Although the existing demulsification methods in the industry have poor efficiency and effect, the traditional demulsification methods have various problems after demulsification, such as the use of different demulsifiers for different vaccines, incomplete demulsification, unclear separation of a water phase and an oil phase, no antigen detection in the water phase and the like. How to find a universal and efficient demulsification method and how to make the antigen in the vaccine enter the water phase become a problem to be solved urgently in the industry.
Chinese patent CN 103088157B mentions that when the foot-and-mouth disease virus RNA is extracted, n-pentanol is added as a demulsifier. However, n-amyl alcohol is used as a demulsifier, so that the efficiency is low, the application is not wide, and antigen components cannot be detected in the water phase of the synthetic peptide vaccine; the mixture of the organic solvent and the acid is used as the demulsifier in the Chinese patent CN106814152A, the demulsifier has various organic solvent types and complex operation, the demulsifier needs vacuum freezing at the temperature of-80 to-50 ℃ for 12 to 18 hours after demulsification, the equipment cost is high, and the separated aqueous phase is at the lower layer and is not convenient to extract.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the problems of high difficulty and irregular data of animal detection and the problems of poor efficiency and effect of the conventional demulsifiers in the quality detection of the conventional animal oil adjuvant vaccine, a preparation method of the demulsifier is provided.
In order to solve the technical problems, the invention adopts the following technical scheme:
a biphasic oil-adjuvanted vaccine demulsifier, comprising chloroform and ammonium sulphate, wherein the volume ratio of chloroform to vaccine emulsion is 2: 1-2, wherein the mass volume ratio of the ammonium sulfate to the vaccine emulsion is 1: 5-20.
The method for demulsifying a demulsifier of a biphasic oil adjuvant vaccine according to claim 1, wherein the demulsifier is added into the biphasic oil adjuvant vaccine, the mixture is shaken and mixed evenly, and then the mixture is centrifuged for 10 to 20 minutes, so that the water phase and the oil phase are completely separated.
The centrifugation conditions were 3000-.
The demulsifier is applied to the antigen recovery of the biphase oil adjuvant vaccine.
The beneficial technical effects of the invention are as follows: the method can effectively and quickly separate the water phase and the oil phase in the dual-phase oil adjuvant vaccine, has short demulsification time, less used reagents, simple operation and low requirement on the operation environment, can be operated at room temperature, separates the water phase into supernatant, has obvious water phase, is convenient for extracting the water phase, and can obviously improve the recovery of the antigen.
Drawings
FIG. 1 is a diagram showing the separation of an aqueous phase layer and an oil phase layer in a demulsification test.
Detailed Description
Because the foot-and-mouth disease virus experiment needs to be operated in a biosafety third-level laboratory, the effect of the demulsifier can be fully displayed by replacing the cat calicivirus with the feline calicivirus with the similar size and character to the foot-and-mouth disease virus.
The following will be described in detail with reference to embodiments, but those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the specific conditions are not noted in the examples, and are performed according to the conventional conditions or conditions suggested by the manufacturer.
Example 1
Preparation of simulated two-phase oil adjuvant vaccine emulsion: 200 ml of feline calicivirus antigen (provided by Zhengzhou Aike biotechnology Co., Ltd., the virus preservation number CCTCC NO: V202003), and 200 ml of ADJ206(W/O/W) Adojia biotechnology Co., Ltd. Emulsifying according to the instruction, and checking the emulsion character to be qualified.
Demulsification test: in a 15 ml centrifuge tube, 5ml of vaccine emulsion was taken, and then 5ml of chloroform, 0.5mg (NH) were added4)2SO4After hand-shaking and uniformly mixing, at room temperature and 4000rpm, centrifuging for 10min, separating the mixed liquid in a centrifuge tube into two layers, namely a water phase layer and an oil phase layer from top to bottom, as shown in figure 1, clearly shown in the figure, the left side is 5ml of emulsion contrast, the centrifuge tube on the right side is 5ml of emulsion, 0.5g of ammonium sulfate and 5ml of chloroform, breaking emulsion and centrifuging, namely, the two layers are separated clearly, the complete separation of the water phase layer of the calicivirus vaccine antigen is realized, the water phase is 2.5ml, the boundary is clear, the distribution is stable, and the water phase is directly taken out at the upper layer.
And extracting the upper layer of the calicivirus antigen aqueous phase layer, detecting the titer of the calicivirus, and determining that the titer of the separated aqueous phase is very close to the titer of the original antigen, so that the fact that the aqueous phase is completely separated out and the virus can be completely recovered can be proved.
Example 2
5ml of emulsified oil adjuvant vaccine is taken to be put into a centrifuge tube, 5ml of chloroform, 0.5g of ammonium sulfate or other inorganic salts which can obviously enhance the polarity of water are added, the mixture is evenly mixed and then centrifuged for 10 minutes in a centrifuge at 4000 revolutions, and the upper layer liquid obtained after centrifugation is the water phase.
Example 3
And (3) taking 10ml of emulsified oil adjuvant vaccine into a centrifuge tube, adding 5ml of chloroform and 0.5g of ammonium sulfate, uniformly mixing, centrifuging for 10 minutes in a centrifuge at 3000 revolutions, and centrifuging to obtain an upper layer liquid, namely a water phase.
Example 4
And (3) taking 10ml of emulsified oil adjuvant vaccine into a centrifuge tube, adding 5ml of chloroform and 2 g of ammonium sulfate, uniformly mixing, centrifuging for 10 minutes in a centrifuge at 3000 revolutions, and centrifuging to obtain an upper layer liquid, namely a water phase.
Example 5
And (3) taking 10ml of emulsified oil adjuvant vaccine into a centrifuge tube, adding 5ml of chloroform and 0.5g of ammonium sulfate, uniformly mixing, centrifuging for 10 minutes in a centrifuge at 3000 revolutions, and centrifuging to obtain an upper layer liquid, namely a water phase.
The recovery volume of the calicivirus aqueous layer and the virus titer data obtained are shown in table 1 below.
TABLE 1
| Experiment number | 1 | 2 | 3 | 4 | 5 | The original aqueous phase |
| Volume of recovered aqueous phase (ml) | 2.4 | 2.4 | 2.5 | 2.4 | 2.5 | 2.5 (theory) |
| Aqueous phase titer TCTD50/ml | 8.5 | 8.67 | 8.32 | 8.72 | 8.53 | 8.5 |
The data for examples 1-5 are shown in Table 1.
In the above examples, ammonium sulfate may be replaced by other inorganic salts such as sodium chloride, sodium acetate, sodium carbonate which significantly enhance the polarity of the water.
Claims (4)
1. A biphasic oil adjuvant vaccine demulsifier, characterized in that: the demulsifier comprises chloroform and ammonium sulfate, and the volume ratio of the chloroform to the vaccine emulsion is 2: 1-2, wherein the mass volume ratio of the ammonium sulfate to the vaccine emulsion is 1: 5-20.
2. The method of demulsifying a biphasic oil-adjuvant vaccine demulsifier of claim 1, wherein: adding the demulsifier into the two-phase oil adjuvant vaccine, oscillating and mixing uniformly, and then centrifuging for 10-20 minutes.
3. The method of demulsifying a biphasic oil-adjuvant vaccine demulsifier of claim 2, wherein: the centrifugation conditions were 3000-.
4. Use of the demulsifier of claim 1 in the recovery of antigen from a biphasic oil-adjuvanted vaccine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111025058.8A CN113680105A (en) | 2021-09-02 | 2021-09-02 | Oil adjuvant vaccine demulsifier and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111025058.8A CN113680105A (en) | 2021-09-02 | 2021-09-02 | Oil adjuvant vaccine demulsifier and application thereof |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202111025058.8A Pending CN113680105A (en) | 2021-09-02 | 2021-09-02 | Oil adjuvant vaccine demulsifier and application thereof |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161789A (en) * | 2007-11-27 | 2008-04-16 | 辽宁大学 | Sand-containing emulsification crude oil demulsification method |
| CN101664549A (en) * | 2009-09-08 | 2010-03-10 | 金宇保灵生物药品有限公司 | Process for purifying antigen of foot and mouth disease vaccine |
| CN102380232A (en) * | 2011-06-30 | 2012-03-21 | 金宇保灵生物药品有限公司 | Emulsion breaking method for aftosa oil emulsion inactivated vaccine |
| CN103215068A (en) * | 2013-05-07 | 2013-07-24 | 武汉凯迪工程技术研究总院有限公司 | Modification method of biomass pyrolysis oil |
| CN104056469A (en) * | 2013-03-18 | 2014-09-24 | 中国石油化工股份有限公司 | Method for biodiesel demulsification |
| CN111060489A (en) * | 2019-12-25 | 2020-04-24 | 安徽中科赛飞尔科技有限公司 | SERS detection method for dazomet or bromadiolone in edible oil |
| CN112587960A (en) * | 2020-11-20 | 2021-04-02 | 天康生物股份有限公司 | Electrolyte demulsifier of missible oil emulsion vaccine, demulsification method and application |
-
2021
- 2021-09-02 CN CN202111025058.8A patent/CN113680105A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161789A (en) * | 2007-11-27 | 2008-04-16 | 辽宁大学 | Sand-containing emulsification crude oil demulsification method |
| CN101664549A (en) * | 2009-09-08 | 2010-03-10 | 金宇保灵生物药品有限公司 | Process for purifying antigen of foot and mouth disease vaccine |
| CN102380232A (en) * | 2011-06-30 | 2012-03-21 | 金宇保灵生物药品有限公司 | Emulsion breaking method for aftosa oil emulsion inactivated vaccine |
| CN104056469A (en) * | 2013-03-18 | 2014-09-24 | 中国石油化工股份有限公司 | Method for biodiesel demulsification |
| CN103215068A (en) * | 2013-05-07 | 2013-07-24 | 武汉凯迪工程技术研究总院有限公司 | Modification method of biomass pyrolysis oil |
| CN111060489A (en) * | 2019-12-25 | 2020-04-24 | 安徽中科赛飞尔科技有限公司 | SERS detection method for dazomet or bromadiolone in edible oil |
| CN112587960A (en) * | 2020-11-20 | 2021-04-02 | 天康生物股份有限公司 | Electrolyte demulsifier of missible oil emulsion vaccine, demulsification method and application |
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Application publication date: 20211123 |