CN106596875B - A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine - Google Patents
A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine Download PDFInfo
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- 229960005486 vaccine Drugs 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 43
- 239000002671 adjuvant Substances 0.000 title claims abstract description 26
- 239000000427 antigen Substances 0.000 claims abstract description 52
- 102000036639 antigens Human genes 0.000 claims abstract description 52
- 108091007433 antigens Proteins 0.000 claims abstract description 52
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 24
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000000746 purification Methods 0.000 claims abstract description 11
- 239000002253 acid Substances 0.000 claims abstract description 10
- 239000003960 organic solvent Substances 0.000 claims abstract description 10
- 235000019441 ethanol Nutrition 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 4
- 238000010828 elution Methods 0.000 claims description 6
- 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
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 abstract description 11
- 238000012360 testing method Methods 0.000 abstract description 10
- 239000012535 impurity Substances 0.000 abstract description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 abstract description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 abstract description 2
- 235000019253 formic acid Nutrition 0.000 abstract description 2
- 230000004044 response Effects 0.000 abstract description 2
- OAYXUHPQHDHDDZ-UHFFFAOYSA-N 2-(2-butoxyethoxy)ethanol Chemical compound CCCCOCCOCCO OAYXUHPQHDHDDZ-UHFFFAOYSA-N 0.000 abstract 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 abstract 1
- 238000004451 qualitative analysis Methods 0.000 abstract 1
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 29
- 230000000052 comparative effect Effects 0.000 description 8
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- 238000011084 recovery Methods 0.000 description 7
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- 239000012895 dilution Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
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- 230000006872 improvement Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000000091 immunopotentiator Effects 0.000 description 2
- 229940023041 peptide vaccine Drugs 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 101100272788 Arabidopsis thaliana BSL3 gene Proteins 0.000 description 1
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
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- 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
- G01N33/15—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 present invention provides a kind of fast qualitative quantitative detecting methods of oil-adjuvant vaccine, and after oil-adjuvant vaccine is demulsified, gained water phase antigen samples carry out ZIPTIP purifying, and sample after purification carries out qualitative and quantitative detection；The breaking method are as follows: oil-adjuvant vaccine is mixed with demulsifier, water phase antigen samples are obtained after layering；The demulsifier includes the mixture of organic solvent and acid, and the organic solvent is selected from one of acetonitrile, n-butanol, benzyl alcohol, butyl diglycol, ethyl alcohol, and the acid is selected from one of hydrochloric acid, acetic acid, formic acid, trifluoroacetic acid.Breaking method provided by the invention can guarantee that antigen is distributed mainly in water phase, and energy actual response goes out the content and quality of Effective Antigens in vaccine；The sample after demulsification is purified using ZIPTIP again, the impurity in sample is effectively removed, substantially increases the purity of sample, sample is thus subjected to qualitative and quantitative analysis again, improve the accuracy and reliability of testing result.
Description
Technical field
The present invention relates to aftosa vaccine detection technique fields, and in particular to a kind of fast qualitative of oil-adjuvant vaccine is quantitative
Detection method.
Background technique
Regulation efficacy test must be tested using this animal in currently available vaccines, existing vaccines quality standard, since country carries out
100% reinforced immunological policy is difficult to select susceptible inspection with animal, and to attack poison high to Experimental Establishment requirement for animal
(BSL3 grades of laboratories), time-consuming (one month or more), capital cost is big.If selecting impressibility using serum neutralization test
Object is technically difficult to the non-susceptible animal for excluding to have cellular immunity, and in inspection, inspection data is not advised frequent occurrence in practice
The problem of rule, influences the accuracy examined.Therefore aftosa vaccine perplexs always in particular for the quality testing of the vaccine of ox
Veterinary drug monitoring department and associated production enterprise.Therefore it needs to develop tested in vitro technology as early as possible, be tried instead of existing animal
It tests.
Current country is just gradually being transitioned into the detection to vaccine endoantigen to inspections of vaccine, and currently more universal side
Method is to be detected after vaccine demulsification to antigen, by the way that vaccine is carried out demulsification processing, is transferred to antigen in water phase, then right
It carries out subsequent detection and analysis.It is well known that vaccine is to be emulsified by antigen and adjuvant with certain proportion, by specific program
, however adjuvant is but that vaccine demulsification detection brings huge obstacle.
Complicated component in the oil adjuvant as used in vaccine emulsion process contains surfactant, immunopotentiator
Equal substances exist, and above-mentioned impurity and demulsifier are often contained in the water phase after demulsification, can not removed in industry above-mentioned
The method of impurity and demulsifier, and impurity and demulsifier etc. can also largely effect on detection process thereafter, cause signal cover or
Interference, has forced down the intensity of antigen signals, or even can not effectively detect antigen therein, due to being randomly assigned for wherein impurity
Property, cause the repeatability of its detection method bad, while increasing instrument maintenance cost.In vaccine industry, how oil is helped
Vaccinating agent carries out demulsification and carries out detection and identification to ingredient in vaccine to be technological difficulties generally acknowledged in the industry, due to oil-adjuvant vaccine
Complicated component, wherein can cause to do to all detection methods containing substances such as a large amount of surfactant and immunopotentiators again
It disturbs, or even can not detect, not can reflect the time of day of vaccine endoantigen.
Although having breaking method in the industry, its efficiency and effect are bad, have kind after traditional breaking method demulsification
Kind problem, such as the problems such as can't detect antigen in incomplete, water phase and oily unintelligible, water phase of mutually demarcating that be demulsified annoying always
Industry.How a kind of efficient breaking method is looked for into urgent problem to be solved in the industry.
ZIPTIP method is a kind of method of desalination before easy mass spectrum, is gone in analysis detection for salts substances
It removes.There has been no the reports that this method is used for antigen purification in the prior art.
Summary of the invention
For the defects in the prior art, the present invention provides a kind of fast qualitative quantitative detection sides of oil-adjuvant vaccine
Method.
The purpose of the present invention is what is be achieved through the following technical solutions:
The present invention provides a kind of fast qualitative quantitative detecting methods of oil-adjuvant vaccine, comprising the following steps:
After oil-adjuvant vaccine is demulsified, gained water phase antigen samples carry out ZIPTIP purifying, and sample after purification is determined
Property quantitative detection；
The demulsifier includes the mixture of organic solvent and acid, and the organic solvent is selected from acetonitrile, n-butanol, benzene first
One of alcohol, butyl diglycol, ethyl alcohol, the acid are selected from one of hydrochloric acid, acetic acid, formic acid, trifluoroacetic acid.Water intaking phase
Through liquid chromatographic detection, rate of recovery of antigen is up to 90% or more.
Preferably, organic solvent is selected from one of acetonitrile, ethyl alcohol in demulsifier, and acid is in trifluoroacetic acid, hydrochloric acid
It is a kind of.
Preferably, 9:1~5:5 is mixed oil-adjuvant vaccine by volume with demulsifier.Demulsifier too high levels can increase
Add the subsequent processing time for being lyophilized or being concentrated to aqueous sample, it is too low, it can not be demulsified.
Preferably, the volume ratio of acid and organic solvent is (0.05~0.5) in the demulsifier: 100.
Preferably, the breaking method further includes that water phase antigen samples are carried out to freeze-drying or concentration to get antigen sample
Product；The condition of the freeze-drying are as follows: 12~18h of vacuum refrigeration at a temperature of -80~-50 DEG C.
Preferably, specific step is as follows for the ZIPTIP purifying:
A1. ZIPTIP is activated using 50%ACN suction；
A2. pipette samples suction is for several times；
A3. TIPTIP is cleaned using the suction of 0.05%TFA water；
A4. the ACN elution samples containing 0.05%TFA are used.
Preferably, the sample after purification is lyophilized or is concentrated.
Preferably, it is detected using the quantitative and qualitative that the method for electrophoresis poststaining carries out antigen samples.
Using breaking method of the invention, due to rate of recovery of antigen height, obtained antigen samples are without being further purified, i.e.,
It can be directly used for the quantitative and qualitative detection of antigen.
The prior art is compared, the present invention have it is following the utility model has the advantages that
1) breaking method demulsification ability provided by the invention is stronger, can guarantee that antigen is distributed mainly in water phase, and water phase
Can be mutually kept completely separate with oily, can actual response go out the content and quality of Effective Antigens in vaccine, rate of recovery of antigen up to 90% with
On.
2) this method uses ZIPTIP purification process, can effectively remove the impurity in antigen samples, pass through specific adsorption
Antigen, removes the impurity such as surfactant in complex sample, at the same the sample obtained after its elution be used directly for it is high-precision
Spend the analysis detection of instrument.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is antigen concentration HPLC test map in water phase after the method demulsification of comparative example 1.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Embodiment 1
The fast qualitative quantitative detecting method for present embodiments providing a kind of oil-adjuvant vaccine, using following steps:
1) sample demulsification will be handled in demulsification as follows:
Vaccine to be checked (commercially available foot and mouth disease vaccine seedling, concentration 75ug/ml) is mixed, the demulsifier with demulsifier
The volume ratio of middle organic solvent selection acetonitrile, acid selection trifluoroacetic acid, acetonitrile and trifluoroacetic acid is 100:0.05.Take 8ml to be checked
Vaccine is placed in centrifuge tube, and vaccine is mixed with demulsifier with volume ratio 8:2, and concussion mixes, and under the conditions of 4 DEG C, is centrifuged with 3000G
15 minutes, lower layer's water phase was carefully extracted with 10ml syringe after centrifugation.
2) the ZIPTIP purifying of sample
Water phase antigen samples made from step 1) are lyophilized or are concentrated by 2.1, are purified using ZIPTIP, step is such as
Under:
1. activating ZIPTIP using 50%ACN suction；
2. pipette samples suction is for several times；
3. cleaning TIPTIP using the suction of 0.05%TFA water；
4. using the ACN elution samples containing 0.05%TFA.
3) qualitative and quantitative detection
It will be 1/10 (i.e. 0.6ml) of water phase after being demulsified by the sample volume of step 2) after purification, i.e. concentration improves 10
Times, electrophoresis poststaining is carried out, its band is analyzed, is compared with its theoretical molecular weight, antigen bands appear in theoretical molecular weight
Size area can be about 7-10ug to its initial characterization, its protein content known to band gray value, and applied sample amount is 10ul sample,
Sample sample concentration is 0.7-1mg/ml thereon, converts volume change (divided by 2) and dilution after its cycles of concentration (divided by 10), demulsification
Effect (divided by 66.67%) its original concentration is about 53-75ug/ml, measures concentration 68.78ug/ml result phase with liquid chromatogram
Symbol.
The present embodiment is compareed using commercially available Schweineseuche synthetic peptide vaccine with theoretical antigen concentration standard, using liquid
Phase chromatography is analyzed.Go out peak position to the HPLC test map sample of antigen concentration theoretical in vaccine to integrate, integrate
Information is as shown in table 1, and antigenic content is embodied in the form of integrated peak areas in sample, integrated peak areas 2738690.To this
The HPLC test map sample of antigen samples concentration goes out peak position and is accumulated in the water phase obtained after the demulsification of embodiment breaking method
Point, integration information is as shown in table 2, and antigenic content is embodied in the form of integrated peak areas in sample, and integrated peak areas is
1674252.It analyzes by contrast, the use of the present embodiment method rate of recovery of antigen is 91.7%, is i.e. demulsification efficiency 91.7%, compared to
Existing method, rate of recovery of antigen greatly improve.
Table 1
Table 2
Note: vaccine is emulsified by adjuvant and antigen, and the volume ratio of adjuvant and antigen is 1:1, therefore the water obtained after being demulsified
Mutually be vaccine volume to be checked 50%, it is miscible with water by demulsifier in this present embodiment, thus after the method for the present invention is demulsified its
Water phase volume has more the volume of demulsifier, i.e. diluting effect compared with vaccine inner aqueous phase, therefore the more theoretical water phase of water phase in the present embodiment
(4ml) increases 50% (2ml), and the integral result for the water phase detection being demulsified in the present embodiment should calculate dilution effect, that is, examines
Integral result is surveyed divided by 66.67%.
Embodiment 2
The fast qualitative quantitative detecting method for present embodiments providing a kind of oil-adjuvant vaccine, using following steps:
1) sample demulsification will be handled in demulsification as follows:
Vaccine to be checked is mixed with demulsifier, organic solvent selects acetonitrile in the demulsifier, and acid selects trifluoroacetic acid, second
The volume ratio of nitrile and trifluoroacetic acid is 100:0.05.5ml vaccine to be checked is taken to mix with demulsifier with volume ratio 5:5, concussion mixes,
Under the conditions of 4 DEG C, with 3000G centrifugation 15 minutes, water phase antigen samples are obtained.
It is demulsified using the method for the present embodiment to aftosa vaccine, rate of recovery of antigen 90.3%.
2) the ZIPTIP purifying of sample
Water phase antigen samples made from step 1) are lyophilized or are concentrated by 2.1, are purified using ZIPTIP, step is such as
Under:
1. activating ZIPTIP using 50%ACN suction；
2. pipette samples suction is for several times；
3. cleaning TIPTIP using the suction of 0.05%TFA water；
4. using the ACN elution samples containing 0.05%TFA.
3) qualitative and quantitative detection
It will be 1/10 (i.e. 0.75ml) of water phase after being demulsified by the sample volume of step 2) after purification, i.e. concentration improves 10
Times, electrophoresis poststaining is carried out, its band is analyzed, is compared with its theoretical molecular weight, antigen bands appear in theoretical molecular weight
Size area can be about 3-5ug to its initial characterization, its protein content known to band gray value, and applied sample amount is 10ul sample,
Loading sample concentration is 0.3-0.5mg/ml, converts volume change (divided by 2) and dilution after its cycles of concentration (divided by 10), demulsification
It is about 45-75ug/ml that effect (divided by 33.33%), which converts its original concentration, measures concentration 67.73ug/ml knot with liquid chromatogram
Fruit is consistent.
Embodiment 3
The fast qualitative quantitative detecting method for present embodiments providing a kind of oil-adjuvant vaccine, using following steps:
1) sample demulsification will be handled in demulsification as follows:
Vaccine to be checked is mixed with demulsifier, in the demulsifier organic solvent select ethyl alcohol, acid select hydrochloric acid, ethyl alcohol with
The volume ratio of hydrochloric acid is 100:0.33, and 7ml vaccine to be checked is taken to mix with demulsifier with volume ratio 7:3, and concussion mixes, in 4 DEG C of items
Under part, with 3000G centrifugation 15 minutes, water phase antigen samples are obtained.
It is demulsified using the method for the present embodiment to aftosa vaccine, demulsification efficiency is 93.5%.
2) the ZIPTIP purifying of sample
Water phase antigen samples made from step 1) are lyophilized or are concentrated by 2.1, are purified using ZIPTIP, step is such as
Under:
1. activating ZIPTIP using 50%ACN suction；
2. pipette samples suction is for several times；
3. cleaning TIPTIP using the suction of 0.05%TFA water；
4. using the ACN elution samples containing 0.05%TFA.
3) qualitative and quantitative detection
It will be 1/10 (i.e. 0.65ml) of water phase after being demulsified by the sample volume of step 2) after purification, i.e. concentration improves 10
Times, electrophoresis poststaining is carried out, its band is analyzed, is compared with its theoretical molecular weight, antigen bands appear in theoretical molecular weight
Size area can be about 7-10ug to its initial characterization, its protein content known to band gray value, and applied sample amount is 10ul sample,
Sample sample concentration is 0.7-1mg/ml thereon, converts volume change (divided by 2) and dilution after its cycles of concentration (divided by 10), demulsification
It is about 65-93ug/ml that effect (divided by 53.85%), which converts its original concentration, measures concentration 70.13ug/ml knot with liquid chromatogram
Fruit is consistent.
Comparative example 1
This comparative example provides a kind of breaking method of aftosa vaccine, essentially identical with the method for embodiment 1, difference
Place is only that: this comparative example demulsifier uses n-butanol.
It is demulsified using the method for this comparative example to aftosa vaccine, the comparative example is using in water phase after n-butanol demulsification
Antigen concentration HPLC test map is as shown in Figure 1, from this figure it can be seen that in antigen theory retention time (23-28min) simultaneously
Antigen is not detected, illustrates that the amount of antigen contained in sample is extremely low, i.e., the rate of recovery of antigen in this comparative example is substantially zeroed, demulsification
Efficiency is substantially zeroed, and demulsification efficiency is 0%.It carries out ZIPTIP after purification, carries out qualitative and quantitative detection, target protein can not be obtained
Band.
The result of comparative example and embodiment explanation, synthetic peptide vaccine use traditional n-butanol breaking method that can not make
For Antigen distribution in water phase, the amount of antigen that can be detected in water phase is extremely low, is not able to satisfy testing requirements, and the method for the present invention is broken
Amount of antigen reaches 90% or more in water phase after cream, meets subsequent purification testing requirements enough.
There are many concrete application approach of the present invention, the above is only a preferred embodiment of the present invention.More than it should be pointed out that
Embodiment is merely to illustrate the present invention, and the protection scope being not intended to restrict the invention.For the common skill of the art
For art personnel, without departing from the principle of the present invention, several improvement can also be made, these improvement also should be regarded as this hair
Bright protection scope.
Claims (4)
1. a kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine, which comprises the following steps:
After oil-adjuvant vaccine is demulsified, gained water phase antigen samples carry out ZIPTIP purifying, and it is qualitative fixed that sample after purification carries out
Amount detection；
The breaking method are as follows: oil-adjuvant vaccine is mixed with demulsifier, water phase antigen samples are obtained after layering；The demulsifier is
The mixture or ethyl alcohol of acetonitrile and trifluoroacetic acid and the mixture of hydrochloric acid；Wherein, the volume ratio of acetonitrile and trifluoroacetic acid is 100:
0.05；The volume ratio of ethyl alcohol and hydrochloric acid is 100:0.33；
9:1~5:5 is mixed oil-adjuvant vaccine by volume with demulsifier；
The volume ratio of acid and organic solvent is 0.05:100 or 0.33:100 in the demulsifier.
2. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 1, which is characterized in that described
Specific step is as follows for ZIPTIP purifying:
A1. ZIPTIP is activated using 50%ACN suction；
A2. pipette samples suction is for several times；
A3. ZIPTIP is cleaned using the suction of 0.05%TFA water；
A4. the ACN elution samples containing 0.05%TFA are used.
3. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 1, which is characterized in that the purifying
Sample afterwards is lyophilized or is concentrated.
4. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 1, which is characterized in that use electrophoresis
The method of poststaining carries out the quantitative and qualitative detection of antigen samples.
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Citations (7)
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| CN105467138B (en) * | 2015-12-04 | 2018-01-26 | 中国农业科学院兰州兽医研究所 | A kind of method for determining aftosa vaccine component and estimating antigenic content |
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| US5505878A (en) * | 1993-08-16 | 1996-04-09 | Exxon Chemical Patents Inc. | Method of demulsifying water-in-oil emulsions |
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