CN109678936A - It is a kind of from aftosa vaccine sample extract 146S antigen devices and methods therefor and application - Google Patents
It is a kind of from aftosa vaccine sample extract 146S antigen devices and methods therefor and application Download PDFInfo
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Abstract
The devices and methods therefor of extraction 146S antigen and application from aftosa vaccine sample that the present invention provides a kind of；The device provided by the invention that 146S antigen is extracted from aftosa vaccine sample, integrated form realizes the extraction 146S antigen from aftosa vaccine sample antigen, and 146S antigen is purified and is concentrated, not only 146S loss is low, the impurity for detecting to antigen and generating interference can also be removed, especially for the removal of nucleic acids in samples impurity, and other existing device or method are unable to reach this effect, the structure and content for keeping its bioactivity of 146S do not change during the extraction process, it repeats to extract 3 testing standard deviations generally only 0.01~0.3 or so, and the 146S antigen obtained can be used for a variety of Antigen Detection Techniques.The monitoring of aftosa vaccine sample quality is carried out to breeding enterprise, production of vaccine enterprise, supervision and management and raising has very important effect.
Description
Technical field
The invention belongs to field of biotechnology, be related to a kind of device that 146S antigen is extracted from aftosa vaccine sample and
Its methods and applications.
Background technique
Aftosa (Foot-and-mouth disease, FMD) is one of strongest disease of domestic animals of infectiousness, disease incidence
100%, which is listed in first of the legal zoonosis reported by International Animal Health tissue (OIE).
Vaccine depends primarily on the content of Effective Antigens in vaccine to the protecting effect of animal.In aftosa vaccine, rise
Effective Antigens to most important protective effect are its intact virus 146S antigen.However, foot and mouth disease virus 146S antigen is very
It is unstable, it easily cracks during production and transportation, the product after cracking not only loses original immunocompetence, Er Qiecheng
For a kind of new impurity, toxic side effect may cause.Therefore the content of intact virus 146S is directly related to vaccine in vaccine
Quality also becomes one of the index that enterprise and Inspection Unit most pay close attention to.Aftosa is gone out in " Chinese veterinary pharmacopoeia " two 〇 First Five-Year Plan year version
The final evaluation criteria of live vaccine quality is animal immune challenge test.This method testing expenses are extremely expensive, annual aftosa
Expense cost of the manufacturing enterprise on animal challenge test is high, and this method detection error is big, there is also the danger of virus diffusion,
Also relate to Animal Experimental Ethical problem.The currently reported method master for being used to detect 146S antigen in aftosa vaccine
Be divided into two classes: the content of measurement 146S antigen analyzes the serotype of virus.Wherein, 146S antigenic content is measured
Method mainly includes sucrose density gradient centrifugation, size exclusion chromatography and ELISA detection method.
The supercentrifugation of sucrose density gradient is the measurement most important method of 146S antigenic content in Past 30 Years.But it should
Method cannot be directly measured when measuring the 146S antigen in aftosa vaccine, and be carried out first to vaccine a series of
Processing, antigen is discharged from vaccine oil emu, and be concentrated or purified.Such as one kind is disclosed in CN102998378B
Removal nucleic acid substances purified virus is precipitated with protamine sulfate first, then further uses PEG6000 concentrating virus, finally again
The method that the virus after concentration is detected using sucrose density gradient ultracentrifugation；It is also disclosed in CN101655452A
Chloroform or trichlorine is added in a kind of method of sucrose density gradient centrifugation detection 146S antigenic content, this method in viral sample
Ethylene mixing is pre-processed, and virus liquid is collected after centrifugation and carries out ultracentrifugal analysis.
Size exclusion chromatography is reported in recent years for analyzing the new technology of foot and mouth disease virus vaccine antigen, party's tool
There are high resolution, fireballing outstanding advantage.Its principle is the area based on 146S antigen size and most of impurity molecule size
Not, the path length difference walked in the chromatography column is to realize separation.Even if molecular weight is different, there is close hydraulic radius
Substance also have similar mobility.Since the production of vaccine technique of different enterprises and horizontal difference, production procedure often relate to
And the impurity such as nucleic acid, lipid, protein of macromolecule, and size exclusion chromatography can not distinguish these substances and 146S, because
This, which needs to pre-process vaccine, can just be measured.Such as CN104634891A discloses a kind of utilization efficient liquid phase size
The method that exclusion chromatography quantitative determines 146S antigenic content in foot-and-mouth disease antigen, wherein to the aftosa vaccine for having been added to adjuvant
It needs to be added n-butanol etc. and carries out demulsification processing, need to be added nuclease removal core to product among not purified aftosa vaccine
Interference of the acid to detection；CN105158130A discloses a kind of method using size exclusion chromatography quantization foot and mouth disease virus,
The processing that middle this method carries out polarity or nonpolar solvent to sample is pre-processed for extracting antigen, or using nuclease
Nucleic acid interference is removed, or is concentrated using PEG precipitating preparation.
The detection method that other utilization immunological method carries out 146S antigen in aftosa vaccine relates equally to first fight
Former separation, purifying, concentration etc..CN105467138B discloses a kind of according to Immune proliferation principle progress aftosa vaccine component
Determine and the method for estimation antigenic content, the step of this method include break milk separation go out antigen phase and be concentrated by ultrafiltration method into
The concentration of row antigen.CN103091490B and CN103076451B discloses a kind of aftosa 146S antigen quantitative ELISA detection examination
Agent box and its application method, wherein finished product vaccine needs that first antigen is sucked out and is detected with ELISA method again with demulsifier demulsification, centrifugation.
To sum up, according to it is existing detection aftosa vaccine in 146S antigen technology, either quantitative analysis either into
The clear type analysis of promoting circulation of blood, is either detected according to sedimentation coefficient or according to molecular dimension, also or according to immunology principle,
One of processing operations such as the extraction to aftosa vaccine antigen, concentration, purifying or a variety of are directed to, normal with realization,
Accurately measurement.
Be related in current existing report the above processing intent method specifically include that using organic solvent etc. by antigen from
It is extracted in vaccine；Foreign protein is precipitated into removal partial impurities using organic solvent；Utilize PEG precipitating or ultrafiltration concentration pair
Antigen is concentrated and is purified；Utilize the nucleic acid impurities etc. in nuclease removal vaccine.The operation that above method is related to includes
Centrifugation, standing, mixing, absorption liquid etc., the vaccine processing method of difference detection is not only very complicated, and (PEG is heavy for operating time length
Form sediment and need to stand overnight), the equipment such as centrifuge are needed, need sample volume big, the multi step strategy of antigen is also possible to lead to epidemic disease
The destruction and loss of 146S antigen, reduce the accuracy of testing result in seedling.
Therefore, a kind of method for how developing new extraction 146S antigen, enables the 146S antigen extracted directly to keep away
Exempt from impurity interference, purity is high, extraction process is easy, may be directly applied to subsequent detection and quantitative analysis, this is for phase critical point
The assessment of fever aphthous vaccine quality is of great significance.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide one kind extracts 146S from aftosa vaccine sample
The devices and methods therefor of antigen and application can be extracted from complicated inactivated foot-and-mouth disease vaccine sample by straightforward procedure
146S antigen, to the detection of 146S antigen, there may be the impurity of interference for removal, realize the concentration of 146S antigen, while keeping 146S
The structure and content of its bioactivity do not change during the extraction process.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
The device of 146S antigen, the dress are extracted in a first aspect, the present invention provides a kind of from aftosa vaccine sample
It sets including purifier and extractor, the purifier is connected to one end of the extractor.
The device provided by the invention that 146S antigen is extracted from aftosa vaccine sample, integrated form are realized from mouth hoof
146S antigen is extracted in epidemic disease vaccine sample antigen, and 146S antigen is purified and is concentrated, and not only 146S loss is low, moreover it is possible to go
Except the impurity for generating interference is detected to antigen, especially for the removal of nucleic acids in samples impurity, and other existing devices or
Method is unable to reach this effect, and the 146S antigen obtained can be used for a variety of Antigen Detection Techniques, such as sucrose density ladder
The methods of degree centrifugation, size exclusion chromatography carry out antigen and quantify, and ELISA, colloid gold immune paper chromatography, immunoprecipitation etc. are immune
The parting and quantitative determination of method progress 146S antigen.In addition, this method is easy to operate, flexible, additional centrifugation is not needed
The equipment such as machine can need flexibly to be used, to raiser, production of vaccine enterprise, vaccine matter according to the different of detection project
The assessment of amount has very important effect.
Preferably, the extractor includes cylindrical container, piston, push rod, first through hole and syringe needle；The cylindrical body holds
One end of device is opening, and the opposite other end is provided with the first through hole, and the syringe needle is connected in the first through hole；Institute
It states push rod to be connected with piston, the push rod and piston are set in cylindrical container.
Preferably, the purifier is connected at the first through hole of the extractor.
Extractor syringe needle provided by the invention is hollow metal needle, and disassembly or group can be carried out in first through hole
Dress.Push rod and piston can move back and forth in cylindrical container.
Preferably, the surface of the cylindrical container is provided with scale.
Preferably, the setting method of the scale are as follows: to be calculated as zero at first through hole, marked to cylindrical container open end
Cylindrical container internal volume.
Preferably, volume representated by every lattice of the scale be 0.05~0.5mL, such as can be 0.05mL,
0.1mL, 0.2mL, 0.3mL, 0.4mL or 0.5mL etc..Every lattice scale is uniformly distributed.
Preferably, the volume of the cylindrical container is 1~10mL.
Preferably, the purifier includes the first column container, gasket and adsorbent, the both ends of first column container
It is provided with the second through-hole and third through-hole, the gasket and adsorbent are set in turn between the second through-hole and third through-hole
Inside one column container.
Second through-hole of purifier can be connected with the first through hole of extractor, and the third through-hole of purifier can be with filter
Fourth hole be connected.The design of purifier can be realized the removal and concentration of impurity.The function is specific by choosing
Adsorbent and adsorption conditions are realized.
Preferably, the adsorbent includes ion exchange resin and/or nonionic exchange adsorption filler.
Preferably, the ion exchange resin is anion exchange resin.
Effect of the anion exchange resin for nucleic acid impurities and pyrolysis product 12S in removal aftosa vaccine sample
Fruit is more excellent.
Preferably, the anion exchange resin includes DEAE Sepharose FF, Q Sepharose FF or ANX
In Sepharose 4FF any one or at least two combination, preferably Q Sepharose FF.
Preferably, the nonionic exchange adsorption filler is the chromatograph packing material with hydrophobic grouping.
The effect that 146S antigen is concentrated in the chromatograph packing material with hydrophobic grouping is more preferable.
Preferably, the chromatograph packing material with hydrophobic grouping includes Butyl Sepharose 4FF and/or Butyl-
650M。
Preferably, described device further includes filter, and the filter includes the second column container and solid film, and described the
Two column container both ends are provided with fourth hole and fifth hole；The solid film be set to fourth hole and fifth hole it
Between.The fourth hole is connected with the first through hole, and the fifth hole is connected with second through-hole.
The purposes of filter be remove aftosa vaccine sample in it is that may be present will affect Antigen Detection Techniques detection
As a result cell fragment, proteins precipitate, and the solid impurity that removal is newly-generated in extraction stages.Filtering of the present invention
Device is usually commercialized 0.1 μm, 0.22 μm or 0.45 μm of syringe needle filter.
Preferably, the solid film is the cellulose acetate with reticular structure.
Preferably, the aperture of the solid film be 100~500nm, such as can be 100nm, 120nm, 150nm,
200nm, 250nm, 300nm, 350nm, 400nm, 450nm or 500nm etc..
The device of rapidly extracting 146S antigen provided by the invention includes three extractor, filter and purifier components,
Wherein filter can be used or not use.The number of each component is unlimited when use, according to the type of vaccine and extracts purpose, can
To carry out the various combination of three components, such as extractor-filter-purifier, extractor-purifier, extractor-purifying
Device-purifier etc..In the present invention, flexibly rank can be realized between extractor, filter and purifier by the through-hole of top and the bottom
It connects, to realize the extraction to 146S antigen, extracts and purify, the different purposes for extracting and concentrating and purifying.
Second aspect is mentioned from aftosa vaccine sample the present invention provides a kind of using device as described in relation to the first aspect
The method for taking 146S antigen, the adsorbent are ion exchange resin, comprising the following steps:
(1) piston of extractor and push rod are pushed into scale is that will be connected to the needle aspirate mouth at first through hole at zero
Fever aphthous vaccine sample and optionally extractant remove syringe needle, stratification after mixing；
(2) the bottom aqueous layer volume after layering is read, then draws 2 × adsorption liquid of same volume, mixing is stood；
(3) 1 × adsorption liquid is drawn using another extractor, is connected to the second through-hole, pushes the push rod of extractor, so that
Adsorption liquid flows out after purifier；
(4) extractor after standing in step (2) is connect with purifier in step (3), by pushing pushing away for extractor
Bar flows out bottom aqueous layer from the third through-hole of purifier, obtains the 146S antigen；
The adsorbent be nonionic exchange adsorption filler, from aftosa vaccine sample extract 146S antigen method with
When adsorbent is ion exchange resin the difference is that: by pushing the push rod of extractor by bottom aqueous layer in step (4)
After the outflow of the third through-hole of purifier, extractor is removed, eluent is drawn using another extractor, pushes pushing away for extractor
Eluent is flowed out by purifier, obtains the 146S antigen by bar.
Extractor is generally mixed by inversion by the method for mixing described in step (1) of the present invention repeatedly.
The purpose of step (3) of the present invention is the surface ligand for balancing adsorption liquid in purifier.
Preferably, the aftosa vaccine sample can be water phase aftosa vaccine sample, be also possible to and adjuvant emulsion
The substance of aftosa vaccine finished product afterwards etc. type.
Water phase aftosa vaccine sample identity is the aqueous phase solution containing 146S antigen；Aftosa vaccine after adjuvant emulsion
Finished product is defined as the lotion formed after mixing the aqueous phase solution containing 146S antigen with oil emulsion adjuvant.
The method provided by the invention that 146S antigen is extracted from aftosa vaccine sample, is optionally added extraction
Agent.May be implemented to after water phase aftosa vaccine sample, emulsification aftosa vaccine finished product and other kinds of aftosa epidemic disease
Seedling sample extracts separation, and the scope of application is wider.Such as when carrying out 146S Antigen extraction to water phase aftosa vaccine sample, both
Extractant can be added and reach removal impurity and other effects to realize the extraction to 146S antigen, also may not need addition extractant
Directly extract；And when carrying out 146S Antigen extraction to the aftosa vaccine finished product after emulsification, addition extractant can reach
Except impurity and other effects to realize the extraction to 146S antigen.
After bottom aqueous layer of the present invention refers to the mixing such as antigen, extractant, the bottom is formed by after stratification
Water phase, wherein antigen is in bottom aqueous layer.
Of the present invention 1 × meaning be normal concentration solution, 2 × meaning be solution concentration be that 1 × solution is dense
2 times of degree.For example, antigen will be contained according to 1:1 if it is desired that the water phase containing antigen finally contains the adsorption liquid of 1 × concentration
Water phase with 2 × adsorption liquid mixed, the adsorption liquid final concentration in such water phase is exactly 1 ×, the as suction of normal concentration
Attached liquid.
It preferably, further include that first through hole is connect with the second through-hole or is led to first after removing syringe needle in step (1)
Hole is connect with fourth hole；, by aftosa vaccine sample after purifier or filter stratification.
Preferably, the time of standing described in step (1) be 0.5~16h, such as can be 0.5h, 1h, 5h, 8h, 10h,
12h, 13h, 15h or 16h etc..
Preferably, the time of standing described in step (1) is 2~40 DEG C, such as can be 2 DEG C, 5 DEG C, 8 DEG C, 10 DEG C, 20
DEG C, 25 DEG C, 30 DEG C, 35 DEG C or 40 DEG C etc..
More specifically, if the temperature stood at room temperature, such as 25 DEG C, 30 DEG C, the time stood at this time compared with
It is short, generally pass through 0.5~4h；If the temperature stood is lower, when such as 2~8 DEG C, the time stood at this time is longer, may
It can be up to for 10 several hours.
Preferably, extractant described in step (1) includes in n-butanol, n-amyl alcohol, n-hexyl alcohol, n-heptanol or n-hexane
Any one or at least two combination.Any one preferably in n-butanol, n-amyl alcohol or n-hexyl alcohol.
In the present invention, when n-butanol, n-amyl alcohol, n-hexyl alcohol are as extractant, the effect of extraction is best.
Preferably, the volume that adsorption liquid is drawn in step (2) is 5~10 times of purifier volume, such as can be 5 times, 6
Again, 7 times, 8 times, 9 times or 10 times etc..
Preferably, the time of standing described in step (2) be 3~10min, such as can be 3min, 4min, 5min,
6min, 7min, 8min, 9min or 10min etc..Preferably 5min.
Preferably, the adsorbent is ion exchange resin, and adsorption liquid described in step (2) is to contain 0.2~0.5M chlorine
Change the PBS solution that the pH value of sodium is 7.5~8.5；The PBS solution that pH value preferably containing 0.3~0.4M sodium chloride is 8.0.
In the present invention, by the measurement and analysis of the electronegativity to 146S, its pyrolysis product 12S and nucleic acid, excellent
Under conditions of the adsorption liquid pH and ionic strength of choosing, separate 146S with other impurity.
In the present invention, choose the solution under above-mentioned condition as adsorbent, for extraction 146S antigen effect most
It is good.Antigen generates loss if concentration in adsorbent if sodium chloride is too low, and interference impurity is difficult to if excessive concentration and is gone
Except clean.Also, optimal adsorbent is the adsorbent with quaternary ammonium group.
Preferably, the adsorbent is nonionic exchange adsorption filler, adsorption liquid described in step (2) be containing 0.5~
The PB solution that the pH value of 2M ammonium sulfate is 7.5~8.5；The PB solution that pH value preferably containing 1M ammonium sulfate is 8.0.
In the present invention, preferred non-ionic adsorbent filler and under the conditions of, 146S antigen can be adsorbed on completely it is non-from
On sub- adsorption stuffing, and being capable of high efficiente callback under the conditions of preferred eluent.At the same time, there is weaker hydrophobic egg
White matter impurity and nucleic acid realize the purification to 146S antigen due to being difficult to be adsorbed on filler simultaneously.
Preferably, the eluent is PB solution.
Preferably as eluent, the concentration of PB solution is 40~60mM, for example, can be 40mM, 45mM, 50mM,
55mM or 60mM etc..
Preferably as eluent, the pH value of PB solution is 7.5~8.0, for example, can be 7.5,7.6,7.7,7.8,
7.9 or 8.0 etc..
It preferably, further include connecting the fourth hole of extractor and filter in step (3) before the second through-hole of connection
Afterwards, it is connected by the fifth hole of filter with the second through-hole of purifier.
It preferably, further include connection filter in step (4) between extractor connection purifier.
It is anti-to extract 146S in the slave aftosa vaccine sample that the present invention provides a kind of as described in second aspect for the third aspect
Application of the former method in the detection of aftosa vaccine sample 146S antigenic content.
It is micro- in 0.2~5 mcg/ml or 2~100 that the method for the invention can be used for handling 146S antigen concentration range
The aftosa vaccine sample of grams per milliliter.Detection low for antigen concentration, needing the antigen of high concentration to carry out, can by it is non-from
The adsorption stuffing of son exchange class is realized first to the extraction of antigen, concentration, then is detected.The present invention is by adjusting extractor processing
Vaccine volume and eluent volume, realize the concentration of different multiples.Such as it is drawn using the extractor that volume is 5mL
The vaccine of 4mL is extracted, final to be extracted using the eluent of 1mL, then vaccine is concentrated about 4 times；It the use of volume is 10mL
Extractor draw the vaccine of 8mL and extracted, finally extracted using 0.5mL eluent, then vaccine is concentrated about 16 times.
Preferably, 146S antigenic content detection method the following steps are included:
(a) 146S antigen is extracted from aftosa vaccine sample using method according to claim 6 or 7, contained
There is the extracting solution of 146S antigen；
(b) in extracting solution described in detecting step (a) 146S antigen content.
Preferably, the detection method of step (b) detection include efficient liquid phase size exclusion chromatography, ELISA method or
Any one in colloid gold reagent block-regulations.
Detection method of the present invention, those skilled in the art can select according to actual needs.For example, efficient liquid phase
The detected value of size exclusion chromatography is the peak area of 146S antigen；ELISA method measurement detected value is the spy that end reaction terminates
The long light absorption value of standing wave；Colloid gold reagent card detected value is the gray value of detection line after reaction；By above measurement, phase is established
Response curve is closed, the content that 146S antigen is finally calculated can be obtained.
Preferably, sodium perchlorate, tyrosine, day are contained in the mobile phase that the efficient liquid phase size exclusion chromatography uses
In aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, arginine, serine, threonine or proline
Any one or at least two combination；Preferably arginine and/or sodium perchlorate.
Preferably, the arginic concentration is 0.1~0.6M, such as can be 0.1M, 0.2M, 0.3M, 0.4M, 0.5M
Or 0.6M etc.；Preferably 0.4M.
Preferably, the concentration of the sodium perchlorate be 0.05~0.4M, such as can be 0.05M, 0.1M, 0.15M,
0.2M, 0.3M, 0.35M or 0.4M etc..
Current existing method extracts 146S antigen for efficient liquid phase ruler from the aftosa vaccine sample after emulsification
Very little exclusion chromatography quantitative analysis 146S antigen.Since there are a large amount of nucleic acid impurity, Wu Fayong in aftosa vaccine sample
Size exclusion chromatography carries out quantitative detection.By means of the present invention, such as extractor-filter-purifier connection side
Formula not only can significantly be eliminated and be extracted efficiently from antigen in extraction in vaccine, and by the purifying of purifier
The interference of 146S antigen amplifying nucleic acid impurity, can accurately carry out quantitative detection.The result of measurement and nuclease digestion, PEG precipitating,
The result that the traditional treatment methods such as ultracentrifugation obtain is compared to more acurrate.The maximum advantage of the present invention and with other technologies not
It is with place, speed is fast and does not need special equipment, such as the centrifuge that PEG precipitating, Ultracentrifugation Method need.It is logical
Cross the extractor of the device to rapidly extracting 146S antigen, three components of filter and purifier be applied in combination and it is specific
Under use condition, flexibly aftosa vaccine sample can be extracted according to properties of samples and detection, in the same dress
Set middle extraction, removal of impurities and the concentration for realizing antigen.
The method of the invention carries out the extraction operation three times of the same vaccine, shows to extract repetition through antigenic content detection
Property is good.
The 146S antigen that the present invention extracts is for the antigen in ELISA method measurement aftosa vaccine sample.The side ELISA
Method measures the serotype and content of 146S antigen, is easy the interference by its pyrolysis product 12S, therefore the extraction of 146S should be wanted
It cannot lead to the destruction of virus and the interference of 12S can be removed.Traditional method such as PEG precipitating and ultracentrifugation can also be real
The removal of existing 12S, but the time is long, needs to stand overnight, and needs the task equipments such as centrifuge even ultracentrifuge, it is difficult to it is real
The quick removal of existing 12S.Make antigen by purifier, antigen is by inhaling by the promotion of extractor using method of the invention
At attached dose, under the conditions of particular solution adsorbent can 12S in adsorption sample without destroying original 146S, and speed it is fast,
The equipment such as centrifuge are not needed.
The 146S antigen gone out that the present invention extracts can be used in colloidal gold antigen detection reagent card measurement antigenic type and estimate
Calculate content.
In the present invention, efficient liquid phase size exclusion chromatography quantitative analysis 146S antigen use aperture be 20~
The hydrophilic modified silica-gel filler of 50nm or the efficient liquid phase size exclusion chromatography post of polymer substrate.Utilize above method optimization
Testing conditions can effectively avoid the absorption of certain vaccine antigens and chromatographic column, improve detection accuracy.
Compared with the existing technology, the invention has the following advantages:
(1) it is provided by the invention from aftosa vaccine sample extract 146S antigen device, integrated form realize from
146S antigen is extracted in aftosa vaccine sample antigen, and 146S antigen is purified and is concentrated, and not only 146S loss is low, also
Energy removal detects the impurity for generating interference to antigen, and especially for the removal of nucleic acids in samples impurity, and existing other fill
It sets or method is unable to reach this effect, the structure of its bioactivity of 146S and content is kept not to become during the extraction process
Change, 3 testing standard deviations of repetition extraction generally only 0.01~0.3 or so, and the 146S antigen obtained can be used for a variety of
Antigen Detection Techniques.
(2) method provided by the invention that 146S antigen is extracted from aftosa vaccine sample, quick, easy to operate, sample
Product dosage is few, flexibility is high, does not need the equipment such as additional centrifuge, and the 146S antigen extracted can be directly used for different technologies
Carry out the detection of 146S antigen.
Detailed description of the invention
Fig. 1 is the device that 146S antigen is extracted in the slave aftosa vaccine finished product of the offer of the embodiment of the present invention 1,1- cylindrical body
Container, 2- piston, 3- push rod, 4- scale, 5- syringe needle, 6- first through hole, 7 solid films, the second column container of 8-, 9- four-way
Hole, 10- fifth hole, 11- adsorbent, 12- gasket, the first column container of 13-, the second through-hole of 14-, 15- third through-hole.
Fig. 2A is after sample A aftosa vaccine finished product is extracted by existing method in the embodiment of the present invention 3 using efficient liquid phase
The map of size exclusion chromatography detection.
Fig. 2 B is after sample B aftosa vaccine finished product is extracted by existing method in the embodiment of the present invention 3 using efficient liquid phase
The map of size exclusion chromatography detection.
Fig. 2 C is after sample C aftosa vaccine finished product is extracted by existing method in the embodiment of the present invention 3 using efficient liquid phase
The map of size exclusion chromatography detection.
Fig. 2 D is after sample D aftosa vaccine finished product is extracted by existing method in the embodiment of the present invention 3 using efficient liquid phase
The map of size exclusion chromatography detection.
Fig. 3 is 146S Antigen extraction steps flow chart schematic diagram in the embodiment of the present invention 4.
Fig. 4 A is to extract sample A 146S antigen and sample C using rapid fractionation method provided by the invention in embodiment 5
The efficient liquid phase size exclusion chromatography map of 146S antigen.
Fig. 4 B is to extract the efficient of sample A 146S antigen and sample C 146S antigen using supercentrifugation in embodiment 5
Liquid phase size exclusion chromatography map.
Fig. 4 C is to extract the efficient of sample A 146S antigen and sample C 146S antigen using the PEG precipitation method in embodiment 5
Liquid phase size exclusion chromatography map.
Fig. 4 D is the efficient liquid for extracting sample A 146S antigen and sample C 146S antigen in embodiment 5 using enzyme digestion
Phase size exclusion chromatography map.
Fig. 5 A is that colloid gold reagent card detects aftosa vaccine finished product 1 × antigen result figure in embodiment 8.
Fig. 5 B is that colloid gold reagent card detects aftosa vaccine finished product 2.5 × antigen result figure in embodiment 8.
Fig. 5 C is that colloid gold reagent card detects aftosa vaccine finished product 5 × antigen result figure in embodiment 8.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1
The present embodiment provides a kind of from aftosa vaccine finished product extracts the device of 146S antigen
The device of 146S antigen is extracted from aftosa vaccine finished product, as shown in Figure 1, including extractor, filter and pure
Change device, extractor includes cylindrical container 1, piston 2, push rod 3, first through hole 6 and syringe needle 5；One end of the cylindrical container
For opening, the opposite other end is provided with first through hole 6, and syringe needle 5 is connected in first through hole 5；The push rod 3 and 2 phase of piston
Even, push rod 3 and piston 2 are set in cylindrical container 1, and the surface of cylindrical container 1 is provided with scale 4；The setting side of scale
Method are as follows: to be calculated as zero at first through hole 6, mark cylindrical container internal volume to 1 open end of cylindrical container；The scale 4
Every lattice representated by volume be 0.5mL；The volume of cylindrical container 1 is 10mL.
Filter includes the second column container 8 and solid film 7, and second column container both ends are provided with fourth hole 9
With fifth hole 10；The solid film is set between fourth hole 9 and fifth hole 10.
Purifier includes the first column container 13, gasket 12 and adsorbent 11, and the both ends of first column container 13 are set
Be equipped with the second through-hole 14 and third through-hole 15, the gasket and adsorbent be set in turn in the second through-hole 14 and third through-hole 15 it
Between the first column container inside.
The fourth hole 9 of filter can be connected with the first through hole 6 of extractor, and the fifth hole 10 can be with purifier
Second through-hole 14 is connected.
Device provided by the invention is not limited only to this kind of connection type of device provided in this embodiment.
Embodiment 2
The present embodiment utilizes the device of the extraction 146S antigen from aftosa vaccine finished product from aftosa vaccine finished product finished product
146S antigen is extracted in oil emu
The extraction of antigen is that the first step of every antigen detection is carried out to aftosa vaccine finished product, it is therefore an objective to remove sample
In hydrophobic components, various adjuvants mainly added, such as ISA206, MF59, mineral oil, tween.Also it can remove
Cell culture impurity such as cell fragment, cell membrane etc..
Extraction step:
1) piston of extractor 2 and push rod 3 are pushed at zero graduation, first through hole 6 is connected with syringe needle 5, draws extraction respectively
The proportional region of 20%~60% aftosa vaccine finished product and extractant for taking body long-pending, vaccine sample and extractant is 1:1
To 12:1, be with 9:1 it is best, extractant be selected from n-butanol, n-amyl alcohol, n-hexyl alcohol, n-heptanol, n-hexane, chloroform；By extractor
Rear syringe needle 5 is mixed by inversion repeatedly to remove, first through hole 6 is engaged on downwards on filter fourth hole 9 (or can also be direct
Will be on the second through-hole 14 of 6 purifier of first through hole), 14h is stood at 4 DEG C, the liquid in extractor is gradually layered；
2) after standing, the scale in extractor, the 146S antigen water phase as extracted are read according to layering interfaces
Volume；
3) by pushing down on the push rod 3 of extractor, the antigen extracted is collected to or is entered processing in next step.
Usually need to be drawn vaccine and extractant respectively with pipettor to one to the extracting operation of aftosa vaccine finished product
It in container, is first centrifuged after mixing, then protrudes into container bottom with pipettor and draw antigen water phase into another container.
In this process, pipettor enters antigen water phase and frequently can lead to the disturbance of layering interfaces and upper layer impurity part is made to enter water
Phase layer generates interference to subsequent detection method.In addition, also complex in operation.
The advantages of carrying out the extraction of antigen using method of the invention is:
1) by the extractor of invention, the difference of aftosa vaccine finished product and extractant designated volume can be easily realized
It draws, while carrying out the hybrid extraction of sample in extractor, stratification, and by the scale on extractor to extracting
Antigen volume record, do not need additional equipment such as pipettor, centrifuge tube etc..
2) it since the density of the hydrophobic components in extractant and vaccine is lower, on the upper layer of antigen phase, therefore pushes down on
The push rod of dynamic extractor is easy to release antigen water phase by the through-hole of extractor bottom, is collected or carries out next step mistake
Filter or purifying cause impurity to enter antigen aqueous layer without the disturbance due to extraction phase interface.
3) volume of extractor is all suitable for the extraction of a small amount of sample and relatively large sample in 1-10mL.
In the present embodiment, commercial available vaccines are extracted by extractor.Respectively with extractor draw 0.9mL vaccine with
Any one in 0.1mL n-butanol, n-amyl alcohol, n-hexyl alcohol, n-heptanol, n-hexane or chloroform, after being operated according to extraction step,
It is stored at room temperature 30min, reads the antigen water phase volume extracted respectively after being layered.The results are shown in Table 1:
Table 1
| Organic solvent | Water phase volume (mL) |
| N-butanol | 0.40 |
| N-amyl alcohol | 0.37 |
| N-hexyl alcohol | 0.35 |
| N-heptanol | 0.25 |
| N-hexane | 0.10 |
| Chloroform | 0.15 |
According to antigen phase volume, the effect of extracting of n-butanol, n-amyl alcohol and n-hexyl alcohol is preferable, and n-hexane and chloroform extract
Take effect poor.
Embodiment 3
The present embodiment uses efficient liquid phase size exclusion chromatography quantitative detection 146S antigenic content
The inactivated foot-and-mouth disease vaccine oil emu finished product for choosing four enterprises respectively is extracted.Sample message is as shown in table 2:
Table 2
| Sample number into spectrum | Inactivated foot-and-mouth disease vaccine oil emu type |
| A | Aftosa is O-shaped, A type, Type Asia 1 tervalence inactivated vaccine |
| B | FMD tye O swine vaccine |
| C | Aftosa is O-shaped, Type Asia 1, A type tervalence inactivated vaccine |
| D | Aftosa is O-shaped, Type Asia 1, A type tervalence inactivated vaccine |
Antigen extraction is carried out to four kinds of vaccines first, in accordance with existing operating method, according to described in CN104892734A
Method detects the antigen extracted, and steps are as follows:
1) extraction of antigen: taking 0.99mL and n-amyl alcohol volume ratio 9:1 to be added in 2mL centrifuge tube four kinds of vaccines respectively,
Sufficiently oscillation, 4 DEG C stand 1 hour, 3000rpm, 4 DEG C, are centrifuged 6min, are slowly inserted into bottom with syringe or liquid-transfering gun and draw bottom
Layer water phase, is transferred in new centrifuge tube, collects bottom antigen water phase.
2) high performance liquid chromatography detection: highly effective liquid phase chromatographic system is purchased from Agilent company, size exclusion chromatography analytical column
TSK G4000SWXLIt is purchased from Tosohaas company；
A) the 50mM phosphoric acid of the 146S antigen sterling sodium sulphate containing 0.1M prepared according to CN104892734A is delayed
Fliud flushing (pH7.2~7.5) is diluted to 60,40,20,10,4,2 or 1 μ g/mL respectively；
B) it is analyzed using highly effective liquid phase chromatographic system and size exclusion chromatography post.Mobile phase: the sodium sulphate containing 0.1M
50mM phosphate buffer (pH7.2~7.5), flow velocity: 0.6mL/min, sample volume 100 μ L, Detection wavelength 259nm.
C) it establishes standard curve: the condition that the antigen after dilution in step a) is respectively adopted in step b) is analyzed,
146S antigen has characteristic absorption peak near 13.4min.The 259nm absorption peak of the 146S antigen of various concentration antigen is accumulated
Get peak area, using peak area as abscissa, the concentration of antigen sterling is that ordinate obtains linear response curve
C=0.0323PA+0.4551
Wherein C is the concentration of 146S antigen, and unit is μ g/mL, and PA is the peak area of absorption peak, unit mAUs.Institute
Obtain the R of standard curve2=0.9991.
D) the 146S antigen extracted is analyzed using the method in step b), 146S antigen property absorption peak is existed
The peak area of 259nm substitutes into the linear response curve in step c), and the antigen concentration extracted is calculated.
In the inactivated foot-and-mouth disease vaccine oil emu of four enterprises of selection, wherein vaccine B, D can be by 146S antigens
Absorption peak is separated with other impurity, detects three times reproducible, and error is small, as shown in Fig. 2A, Fig. 2 B, Fig. 2 C and Fig. 2 D；And epidemic disease
There is the absorption peak that a large amount of impurity cover 146S antigen in seedling A, D, can not accurately detect.Therefore, with common extraction antigen
Method, commercially available part vaccine can not be measured normally, this just brings the supervision of enterprise, supervision department
Problem.Antigen extraction is carried out to aftosa vaccine finished product using the present invention, can solve the above problem.
Embodiment 4
The present embodiment utilizes device and method provided by the invention, measures the influence of purifier treatment conditions
For interference problem is detected present in embodiment 3, key is that the extraction of antigen needs to remove and generates interference
Substance.In the present invention, which realized by the function of purifier.The present embodiment is described by taking vaccine A as an example, the present invention
The treatment conditions of purifier how to influence the property and testing result of the 146S antigen extracted.
Adsorbent uses ion exchange resin.It is as shown in Figure 3 to extract flow diagram for Antigen extraction step:
1) piston 2 of 2.5mL extractor and push rod 3 are pushed at zero graduation, first through hole 6 is connected with syringe needle 5, inhales respectively
Take 1.35mL vaccine and 0.15mL n-amyl alcohol；It is quiet at 4 DEG C with 5 downwardly direction of syringe needle after extractor is mixed by inversion repeatedly
The liquid set in 30min device to be extracted is gradually layered；
2) volume for reading bottom aqueous layer antigen, 2 × adsorption liquid of same volume is drawn with 5 downwardly direction of syringe needle, is mixed
5min is stored at room temperature after conjunction；Adsorption liquid is that the PBS that the pH value containing different sodium chloride concentrations (specific concentration is shown in Table 3) is 8.0 is molten
Liquid；
3) 1 × adsorption liquid that 8 times of volumes of purifier are drawn using second extractor, is attached to the fourth hole of filter
9 and the second through-hole 14 of purifier the purified device bottom of adsorption liquid is flowed out by pushing down on the push rod 3 of extractor, put down
The surface ligand of adsorbent in weighing apparatus purifier；
4) extractor in step 3) is connected by extractor-filter-purifier direction, by pushing down on extraction
The push rod 3 of device slowly flows the aqueous phase solution that bottom contains antigen through filter, purifier from purifier third through-hole 15
Out, the 146S antigen as extracted.
It is carried out according to antigen of the efficient liquid phase size exclusion chromatography detecting step in embodiment 3 to the vaccine A extracted
Measurement.Due to being diluted in extractor by 2 × adsorption liquid, last measurement concentration need to be multiplied by 2.
The influence for investigating the concentration of the sodium chloride of adsorption liquid and the type of adsorbent respectively, as a result such as table 3.
Table 3
The treatment conditions of purifier will greatly affect the dense of the 146S antigen extracted it can be seen from the result of table 3
Measurement result is spent, the sodium chloride concentration of adsorption liquid is too low, and antigen has loss, and it is excessively high, it is difficult to an interference impurity removal completely, it must
It need be in optimal range.According to measurement result, the concentration of the chlorination processes sodium of optimal adsorption in 0.2~0.5M, preferably 0.3~
0.4M；Optimal adsorbent is the adsorbent with quaternary ammonium group group.
Embodiment 5
It is high to removal that the present embodiment tests the 146S antigen that Antigen extraction method of the invention obtains and existing extracting method
The effect of effect liquid phase size exclusion chromatography detection interference impurity compares.It chooses vaccine A and vaccine C in embodiment 3 and carries out effect ratio
Compared with.
1. rapid fractionation method of the invention: carrying out the extraction of antigen according to step described in embodiment 4, wherein adsorbent
With Q Sepharose FF, adsorption liquid sodium chloride concentration is 0.3M.
The 2.PEG precipitation method: first according to the Antigen extraction method in embodiment 3,146S is extracted from vaccine；In water phase
PEG6000 and 0~0.5M sodium chloride of final concentration 6-8% (w/v) are added in 146S antigen, mixes dissolution completely, it is placed in 2~
After standing 10~16h at 8 DEG C, 10000rpm is centrifuged 10min, surveys after discarding supernatant with the pH7.5 with water phase antigen same volume
~8.2 PBS buffer solution measures after redissolving.
3. supercentrifugation: first according to the Antigen extraction method in embodiment 4,146S is extracted from vaccine；By A, C
The antigen phase collected after demulsification takes 10mL to add in 10mL ultracentrifugation pipe respectively, using 20000~50000rpm centrifugation 2~
5h, 2~8 DEG C of temperature；It is redissolved with the PBS of 10mL pH 7.5~8.2 to original volume and is measured after discarding supernatant.
4. enzyme digestion: first according to the Antigen extraction method in embodiment 4,146S is extracted from vaccine；It will demulsification
The nuclease that 20~200U/mL is added in vaccine sample A and C, measures 146S antigenic content after 4~25 DEG C of 2~16h of placement.
Four kinds of resulting optimal results of method are compared, table 4 is as a result summarized in.
Table 4
Fig. 4 A, Fig. 4 B, Fig. 4 C and Fig. 4 D are Different Extraction Method to 146S antigen testing result map.It can by testing result
Know, mainly to high performance liquid chromatography detection generate interference be nucleic acid impurity, be completely removed after enzymic digestion, and exceed the speed limit from
Heart method is difficult to completely remove this kind of nucleic acid chaff interferent, causes measurement result relatively low.Extracting method testing result of the invention and enzyme
The result of digestion is completely the same, shows that interference impurity can not only be removed, and result is accurate, not will cause the loss of antigen.
For the angle of operation, method of the invention does not need other equipment, and other methods generally require centrifuge；In processing
Between in upper and treating capacity also with advantage.
Embodiment 6
The present embodiment tests the repeatability of extracting method of the invention
The aftosa vaccine finished product for choosing different company source on the market carries out antigen according to the step in embodiment 4
Extraction, and with the efficient liquid phase size exclusion chromatography detection method in embodiment 3 carry out three times repeat extract and measurement, as a result see
Table 5.Table 5 show to detect three times it is reproducible, and to the sample of various serotype, different company source can be carried out extraction and
Subsequent measurements.
Table 5
Embodiment 7
The present embodiment uses ELISA detection kit Detection and Extraction antigen
The principle of ELISA measurement aftosa vaccine finished product are as follows: it is coated with microwell plate with the preparation of one plant of monoclonal antibody, in addition one
Strain monoclonal antibody prepares HRP enzyme marker.When containing antigen in sample, antibody-antigen-antibody-enzyme compound is formed,
It develops the color when substrate is added, different colours is shown according to the amount of antigen.But since 146S and its pyrolysis product 12S contain
There is antigenic determinant, therefore can be difficult to determine the content of Effective Antigens 146S in vaccine in conjunction with antibody.The present embodiment
Description can remove antigen lysate 12S present in sample using method extraction 146S of the invention and do to ELISA measurement
It disturbs.
The electronegativity of 146S antigen and 12S under condition of different pH are analyzed, as a result such as table 6.As shown in Table 6,146S
Pyrolysis product 12S there is significantly stronger elecrtonegativity, using this point, point of 146S and 12S may be implemented under given conditions
From.
Table 6
| 146S | 12S | |
| pH 7.0 | -9.86±0.04 | -14.0±0.63 |
| pH 8.0 | -10.9±0.80 | -19.5±1.29 |
| pH 9.0 | -12.6±0.40 | -23.1±2.47 |
Vaccine A is placed 7 days at 45 DEG C, cracks 146S antigen therein completely.Respectively by after heating vaccine A with
Not heated vaccine A is according to extracting method (step traditional in rapid fractionation method and embodiment 3 of the invention in embodiment 6
1) operation carries out the extraction of antigen, reuses the measurement that commercially available foot-and-mouth disease antigen ELISA detection kit carries out antigen.As a result
Compare as shown in table 7:
Table 7
It is in contrast table 7 as a result, vaccine is extracted with traditional extraction method, even if the 146S in vaccine has been cracked into 12S, still
It can be detected that there is similar testing result with unheated vaccine by ELISA；And extracted with method of the invention,
ELISA measurement result also has been approached PBS control group under higher extension rate in vaccine A after heating cracking, shows wherein
12S can largely be removed, reduce vaccine present in 12S interference.
Embodiment 8
The present embodiment detects aftosa vaccine finished product using colloid gold reagent card
The aftosa vaccine finished product extremely low to concentration, it is difficult to detect antigen serotype therein with colloid gold reagent card.This
Embodiment utilizes method of the invention, realizes concentration, while Antigen extraction so as to carry out antigen detection.
The Antigen extraction method according to embodiment 4 is chosen, the O-shaped sample for measuring antigen concentration in 0.2~5 μ g/mL carries out
The extraction of antigen.Steps are as follows:
1) piston 2 of 2.5mL extractor and push rod 3 being pushed at zero graduation respectively, first through hole 6 is connected with syringe needle 5, point
It Xi Qu not 0.9mL vaccine and 0.1mL n-amyl alcohol；
2) it after being mixed by inversion extractor repeatedly, is stood in 30min device to be extracted at 4 DEG C with 5 downwardly direction of syringe needle
Liquid be gradually layered；
3) volume for reading bottom aqueous layer antigen, 2 × adsorption liquid of same volume is drawn with 5 downwardly direction of syringe needle, is mixed
5min is stored at room temperature after conjunction；Adsorption liquid is the PB solution that the pH value of 1M ammonium sulfate is 8.0；
4) balance of purifier: 1 × adsorption liquid of 5 times of volumes of purifier is drawn using second extractor, is attached to pure
Change the second through-hole of device 14, by pushing down on the push rod 3 of extractor, the purified device bottom of adsorption liquid is flowed out, purifier is balanced
The surface ligand of middle adsorbent；
5) extractor in step 3) is connected by extractor-filter-purifier direction, by pushing down on extraction
The push rod 3 of device slowly flows the aqueous phase solution that bottom contains antigen through filter, purifier from purifier third through-hole 15
Out, extractor is removed；
6) eluent identical with water phase volume in step 3) is drawn using third extractor, linking is on the filter
End, by pushing down on the push rod 3 of extractor, by eluent through filter, purifier slowly from purifier third through-hole 15
It releases, trickle is the 146S antigen extracted；Eluent is 50mM, and pH value is the PB solution of 7.5-8.0.
Respectively by the extractor volume in step 1), vaccine volume is corresponding to n-butanol volume expands, and in step 6)
Effluent volume is constant, it can be achieved that distinguishing antigen 2.5 times and 5 times of concentration.For example, 2.25mL epidemic disease is drawn in step 1) respectively
Seedling and 0.25mL n-amyl alcohol, and the effluent volume in step 6) is constant, then the antigen concentration 2.5 finally extracted ×；By step
1) the extractor volume in is changed to 10mL, respectively absorption 4.5mL vaccine and 0.5mL n-amyl alcohol, and the eluting liquid in step 6)
Product is constant, then and the antigen concentration 5 finally extracted ×.It is extracted from higher volume of vaccine by using higher volume of extractor
Antigen, and with the elution antigen of less volume, bigger cycles of concentration can be obtained.
The 50 μ L of antigen extracted is taken respectively, is added dropwise in commercially available antigen colloidal gold reagent card hole, is observed after 20min,
As a result as shown in Fig. 5 A, Fig. 5 B and Fig. 5 C, showing that C line occur in all test cards, detection is effective, and 1 × antigen concentration is low,
T line is not significant on O-shaped detection reagent card, and be concentrated 2.5 × and 5 × antigen test card in there is T line；All samples are in A type
It is not significant on detection reagent card.This is the result shows that can be used for the detection of low concentration sample using this method.
Embodiment 9
The present embodiment extracts 146S antigen using method provided by the invention, to high performance liquid chromatography size exclusion chromatography
Mobile phase condition is investigated.
Table 8
When carrying out the analysis of 146S antigen using efficient liquid phase size exclusion chromatography, key is antigen in analytic process
In should not have excessive non-specific interaction with chromatographic column, lead to the absorption of antigen.According to mobile phases different in table 8
Under the conditions of, when the concentration of sodium sulphate gradually increases, the lag of retention time is all had occurred in the antigen of two kinds of vaccine products, is shown
Due to the excessively high hydrophobic interaction that can enhance 146S antigen Yu chromatography molecular columns of salinity.However the aftosa epidemic disease of certain enterprises
Seedling sample may have stronger phase interaction due to the particularity of antigen amino acid sequence under certain analysis conditions with chromatographic column
With influencing testing result.Such as the vaccine 2 in the present embodiment only has very low absorption peak under the conditions of multiple mobile phases.It is right
Result above is analyzed, and salinity enhancing or reduction cannot all significantly improve its absorption behavior, shows that the 146S in vaccine 2 is anti-
Former non-specific adsorption is not only due electrostatic interaction or hydrophobic effect, and may be hydrogen bond.Shape is removed by being added to have
At the amino acid of free amine group, hydroxyl other than peptide bond, to be saturated the adsorption site on chromatographic column surface, it may be possible to shield antigen
With the non-specific binding of chromatographic column.It can be seen that from the 5-11 group result of table 8, it, can when certain density amino acid is added
Effectively to weaken the absorption of 146S antigen, the peak area at detection peak is improved, the lag of retention time is avoided.Preferred condition is,
0.4M arginine is added in mobile phase.In another embodiment, tyrosine, aspartic acid, histidine, day are added in mobile phase
One of winter amide, glutamic acid, lysine, glutamine, arginine, serine, threonine, proline.In addition, perchloric acid
Sodium also shows the property that inhibition 146S antigen outstanding adsorbs on a column.Sodium perchlorate has the liberation characteristic of salt simultaneously
With the hydrophobicity of perchlorate's group, it is thus possible at the same can weaken 146S antigen and chromatographic column Electrostatic Absorption and hydrophobic work
Keep testing result more acurrate to reduce the loss of 146S antigen in the detection with absorption.
Embodiment 10
The present embodiment is to the Antigen extraction in water phase aftosa vaccine
The supernatant, vaccine of infection cell culture may have in the 146S antigen aqueous solution before purification or in purification process
The substances such as part cell fragment, lipid.When extracting antigen by the methods of ultracentrifugation, PEG precipitating, these impurity can also lead to
Centrifugation is crossed to be collected and can not separate with 146S antigen well.Method in through the invention can also be realized in this kind of vaccine
Finished product detected before extraction, remove this partial impurities.Water phase aftosa vaccine sample and the newborn vaccine finished product of aftosa oil
Antigen extraction difference is that, due to not containing oil emulsion adjuvant, extraction step is not required.Its extracting method can be according to embodiment 4
Or the method in 9 carries out, also may not need embodiment 4 or the step 1) in 9 original methods and step 2) can carry out mentioning for antigen
It takes.
The present invention equally includes other combinations of extraction element, such as passes through the purifier for ionic adsorbent of connecting
And the purifier of non-ionic adsorbent realizes the removal of nucleic acid impurities and the concentration of antigen simultaneously；In extraction stages, extraction is utilized
It takes device to draw the processing of antigen water phase etc. that other materials such as nuclease is extracted to operate.
It of the invention is extracted from aftosa vaccine sample the Applicant declares that the present invention is explained by the above embodiments
The devices and methods therefor of 146S antigen and application, but the invention is not limited to above-mentioned processing step, that is, do not mean that the present invention
Above-mentioned processing step, which must be relied on, to be implemented.It should be clear to those skilled in the art, any changes to of the invention
Into addition, selection of concrete mode of equivalence replacement and auxiliary element to raw material selected by the present invention etc. all fall within the present invention
Protection scope and the open scope within.
Claims (10)
1. a kind of device for extracting 146S antigen from aftosa vaccine sample, which is characterized in that described device includes purifier
And extractor；The purifier is connected to one end of the extractor.
2. the apparatus according to claim 1, which is characterized in that the extractor include cylindrical container, piston, push rod,
First through hole and syringe needle；One end of the cylindrical container is opening, and the opposite other end is provided with the first through hole, described
Syringe needle is connected in the first through hole；The push rod is connected with piston, and the push rod and piston are set in cylindrical container；
Preferably, the purifier is connected at the first through hole of the extractor.
3. the apparatus of claim 2, which is characterized in that the surface of the cylindrical container is provided with scale；
Preferably, the setting method of the scale are as follows: to be calculated as zero at first through hole, mark cylinder to cylindrical container open end
Body container internal volume；
Preferably, volume representated by every lattice of the scale is 0.05~0.5mL；
Preferably, the volume of the cylindrical container is 1~10mL.
4. device according to any one of claim 1-3, which is characterized in that the purifier holds including the first cylinder
Device, gasket and adsorbent, the both ends of first column container are provided with the second through-hole and third through-hole, the gasket and absorption
Agent is set in turn in inside the first column container between the second through-hole and third through-hole；
Preferably, the adsorbent includes ion exchange resin and/or nonionic exchange adsorption filler；
Preferably, the ion exchange resin is anion exchange resin；
Preferably, the anion exchange resin includes DEAE Sepharose FF, Q Sepharose FF or ANX
In Sepharose 4FF any one or at least two combination, preferably Q Sepharose FF；
Preferably, the nonionic exchange adsorption filler is the chromatograph packing material with hydrophobic grouping；
Preferably, the chromatograph packing material with hydrophobic grouping includes Butyl Sepharose 4FF and/or Butyl-650M.
5. device described in any one of -4 according to claim 1, which is characterized in that described device further includes filter, described
Filter includes the second column container and solid film, and second column container both ends are provided with fourth hole and fifth hole；
The solid film is set between fourth hole and fifth hole；The fourth hole is connected with the first through hole, described
Fifth hole is connected with second through-hole；
Preferably, the solid film is the cellulose acetate with reticular structure；
Preferably, the aperture of the solid film is 100~500nm.
6. a kind of extract 146S antigen using device according to any one of claims 1 to 5 from aftosa vaccine sample
Method, which is characterized in that the adsorbent is ion exchange resin, comprising the following steps:
(1) piston of extractor and push rod are pushed into scale is that will be connected to the needle aspirate aftosa at first through hole at zero
Vaccine sample and optionally extractant remove syringe needle, stratification after mixing；
(2) the bottom aqueous layer volume after layering is read, then draws 2 × adsorption liquid of same volume, mixing is stood；
(3) 1 × adsorption liquid is drawn using another extractor, is connected to the second through-hole, pushes the push rod of extractor, so that absorption
Liquid flows out after purifier；
(4) extractor after standing in step (2) is connect with purifier in step (3), by pushing the push rod of extractor will
Bottom aqueous layer is flowed out from the third through-hole of purifier, obtains the 146S antigen；
The adsorbent is nonionic exchange adsorption filler, and the method and absorption of 146S antigen are extracted from aftosa vaccine sample
When agent is ion exchange resin the difference is that: by pushing the push rod of extractor by bottom aqueous layer from pure in step (4)
After the third through-hole outflow for changing device, extractor is removed, eluent is drawn using another extractor, pushes the push rod of extractor,
Eluent is flowed out by purifier, obtains the 146S antigen.
7. according to the method described in claim 6, it is characterized in that, the aftosa vaccine sample includes water phase aftosa vaccine
Aftosa vaccine finished product after sample and/or emulsification；
Preferably, after removing syringe needle in step (1), further include first through hole is connect with the second through-hole or by first through hole with
Fourth hole connection, by aftosa vaccine sample after purifier or filter stratification；
Preferably, the time of standing described in step (1) is 0.5~16h；
Preferably, the time of standing described in step (1) is 2~40 DEG C；
Preferably, extractant described in step (1) includes any in n-butanol, n-amyl alcohol, n-hexyl alcohol, n-heptanol or n-hexane
It is a kind of or at least two combination；Any one preferably in n-butanol, n-amyl alcohol or n-hexyl alcohol；
Preferably, the volume that adsorption liquid is drawn in step (2) is 5~10 times of purifier volume；
Preferably, the time of standing described in step (2) is 3~10min；Preferably 5min；
Preferably, the adsorbent is ion exchange resin, and adsorption liquid described in step (2) is to contain 0.2~0.5M sodium chloride
PH value be 7.5~8.5 PBS solution；The PBS solution that pH value preferably containing 0.3~0.4M sodium chloride is 8.0；
Preferably, the adsorbent is nonionic exchange adsorption filler, and adsorption liquid described in step (2) is to contain 0.5~2M sulphur
The PB solution that the pH value of sour ammonium is 7.5~8.5；The PB solution that pH value preferably containing 1M ammonium sulfate is 8.0；
Preferably, the eluent is PB solution；
Preferably, the concentration of the PB solution is 40~60mM；
Preferably, the pH value of the PB solution is 7.5~8.0；
Preferably, in step (3) before the second through-hole of connection, further include by after the connection of the fourth hole of extractor and filter,
It is connected by the fifth hole of filter with the second through-hole of purifier；
It preferably, further include connection filter in step (4) between extractor connection purifier.
8. the method for 146S antigen according to claim 6 or 7 of extracting from aftosa vaccine sample is in aftosa vaccine
Application in the detection of sample 146S antigenic content.
9. application according to claim 8, which is characterized in that the method for the 146S antigenic content detection includes following step
It is rapid:
(a) 146S antigen is extracted from aftosa vaccine sample using method according to claim 6 or 7, contained
The extracting solution of 146S antigen；
(b) in extracting solution described in detecting step (a) 146S antigen content；
Preferably, the detection method of step (b) detection includes efficient liquid phase size exclusion chromatography, ELISA method or colloid
Any one in gold reagent block-regulations.
10. application according to claim 9, which is characterized in that the stream that the efficient liquid phase size exclusion chromatography uses
Contain sodium perchlorate, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, essence in dynamic phase
In propylhomoserin, serine, threonine or proline any one or at least two combination；Preferably arginine and/or high chlorine
Sour sodium；
Preferably, the arginic concentration is 0.1~0.6M；Preferably 0.4M；
Preferably, the concentration of the sodium perchlorate is 0.05~0.4M.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112034028A (en) * | 2020-08-25 | 2020-12-04 | 中国科学院过程工程研究所 | Method for detecting 146S antigen in foot-and-mouth disease vaccine based on capillary electrophoresis method and application thereof |
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| 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 |
| CN104818254A (en) * | 2015-05-06 | 2015-08-05 | 中国科学院过程工程研究所 | Method of purifying foot-and-mouth disease inactivated virus antigen through ion exchange chromatography |
| CN104892734A (en) * | 2015-04-30 | 2015-09-09 | 中国科学院过程工程研究所 | Method for purifying foot-and-mouth disease inactive virus antigen through hydrophobic interaction chromatography |
| CN108469466A (en) * | 2018-03-23 | 2018-08-31 | 中国检验检疫科学研究院 | A kind of on-line extraction purification needle point electrospray ionization device and sample fast determining method |
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| 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 |
| CN104892734A (en) * | 2015-04-30 | 2015-09-09 | 中国科学院过程工程研究所 | Method for purifying foot-and-mouth disease inactive virus antigen through hydrophobic interaction chromatography |
| CN104818254A (en) * | 2015-05-06 | 2015-08-05 | 中国科学院过程工程研究所 | Method of purifying foot-and-mouth disease inactivated virus antigen through ion exchange chromatography |
| CN108469466A (en) * | 2018-03-23 | 2018-08-31 | 中国检验检疫科学研究院 | A kind of on-line extraction purification needle point electrospray ionization device and sample fast determining method |
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| CN112034028A (en) * | 2020-08-25 | 2020-12-04 | 中国科学院过程工程研究所 | Method for detecting 146S antigen in foot-and-mouth disease vaccine based on capillary electrophoresis method and application thereof |
| CN112034028B (en) * | 2020-08-25 | 2021-10-15 | 中国科学院过程工程研究所 | Method for detecting 146S antigen in foot-and-mouth disease vaccine based on capillary electrophoresis method and application thereof |
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