CN103937757B - A kind of Latex agglutination test purification process - Google Patents
A kind of Latex agglutination test purification process Download PDFInfo
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Abstract
The invention provides a kind of Latex agglutination test (JEV) purification process, the method comprehensively have employed microfiltration clarification method, and method of purification is concentrated by ultrafiltration, and repeats the series of process such as filter wash method, increases the organic efficiency of JEV to greatest extent, reduce purification cost.It is particularly suited for preparing vaccine with Latex agglutination test concentrated solution finished product prepared by the present invention, vaccine prepared by the Latex agglutination test concentrated solution finished product prepared with the present invention is for the Latex agglutination test vaccine of prior art, there is high purity, safety is high, the advantage such as homogeneity is good, good immune effect.Meanwhile, present invention process is easy, cost is relatively low, has prominent scale application prospect.
Description
Technical field
The present invention relates to a kind of viral purification methods, particularly relate to a kind of Latex agglutination test purification process.
Background technology
Epidemic encephalitis type B is by flaviviridae (Flaviviridae) Flavivirus (Flavivirus) Japan second
The disease of a kind of Zoonosis that type encephalitis (Japanese Encephalitis Virus, JEV) causes.Pig quilt
It is considered the most important animal that accrues of primary disease, farrowing sow can be caused to miscarry, stillborn fetus, mummy tire,
Boar generation orchitis；Clinically with high heat, disturbance of consciousness, tic, respiratory failure and meningeal irritation sign as spy
Levy.Pig japanese b encephalitis is popular not only causes serious harm to pig industry, but also serious threat human health.
Vaccine is one of main method preventing this disease.At present, the pig japanese b encephalitis vaccine of Chinese commodity is main
It is Mus brain inactivated vaccine and attenuated vaccine.Wherein based on attenuated vaccine, its safety and effectiveness are mainly by disease
The impact of the poison factor such as titre and the pure property of antigen.If commercialized vaccine directly enters with the virus liquid of cell proliferation
The shadow of the impurity such as the production of row finished product Seedling, can be by cell breakage product, medium component, products of cellular metabolism
Ring, the side effect such as anaphylaxis, exothermic reaction easily occur after causing vaccine immunity animal.So, improve virus
Titre and the pure property of antigen are an up vaccine quality Basic Ways.
Doughnut membrane filtration technique belongs to tangential flow filtration technology (Tangential Flow Filtration, TFF)
Category, also known as cross flow filter (Cross-Flow Filtration, CFF): feed liquid with certain flow velocity at film
Upper surface circulation, through film to passing through end, and can be more than the material meeting quilt of membrane aperture less than the material of membrane aperture
Film retains, thus realizes the concentration of target substance and the fractionated of different material.
Comparing with traditional Flat Membrane bag, hollow fiber column has the open flow passage structure of fiber tubulose, without sieve
The tubulose flow passage structure of net avoids the random high turbulences of feed liquid, therefore has lower shearing force, gentle
Operation can effectively prevent coming off and the gathering of albumen of viral surface glycoprotein, be conducive to the complete of protection virus
Whole property, prevents virion from contributing to passing through and removing of foreign protein while assembling.
Doughnut clarification and concentration technology currently for Latex agglutination test there is no report.
Summary of the invention
Present invention seek to address that the technology such as poor, the immune effect difference of existing Latex agglutination test vaccine homogeneity are asked
Topic, it is provided that a kind of Latex agglutination test using microfiltration clarification system and purification system realization being concentrated by ultrafiltration
Purification process.
For realizing above technical purpose, the present invention by the following technical solutions:
A kind of Latex agglutination test purification process, the method is by microfiltration clarification system and is concentrated by ultrafiltration pure
Change system realizes, and process is as follows:
The assembling of 1 system
Under aseptic condition, microfiltration clarification system and ultrafiltration concentration purification system are carried out group according to assembling requirement
Dress.Aseptic 0.45 μm or 0.65 μm, intact doughnut can be installed in microfiltration clarification system
Micro-filtration membrane, can install aseptic 100KD or 300KD, intact hollow in purification system is concentrated by ultrafiltration
Fiber ultrafiltration membrane.
2 system integrity detections
Pressure keeps the integrity of method detecting system.
The process of 3 systems
3.1 clean and sterilizing
0.5M NaOH solution is filled the head tank of system, immersion treatment 20min, ON cycle pump
300rpm, carries out cleaning and sterilization treatment 30min of system.
3.2 washings and flux detection
After sterilizing terminates, drain intrasystem NaOH solution.Aseptic ultra-pure water is filled the head tank of system,
ON cycle pump, 300rpm circulates 30min, abandons the most intrasystem liquid, the most repeatedly wash, until being
In system, PH is about 7.0.
3.3PBS process
After washing terminates, abandon last to the greatest extent ultra-pure water.0.1M PBS solution is filled head tank, and unlatching follows
Ring pump 300rpm circulation flushing 20min.
In technical scheme described above, the NaOH solution used in step 3.1 and 3.2 plays cleaning, sterilizing
Effect, it is possible to select the alcoholic solution of 50%～80%, or use the mode of steam sterilization to carry out system sterilizing.
A kind of Latex agglutination test purification process, the method is by microfiltration clarification system and is concentrated by ultrafiltration pure
Change system realizes, and the method comprises the following steps:
1) aseptic polysorbas20 is added in Latex agglutination test liquid in the ratio of 0.5%～10% (v/v), vibration
Process；
2) virus liquid after step 1) oscillation treatment is injected in the head tank of microfiltration clarification system, open
Open circulating pump, through 0.45 or 0.65 μm doughnut microfiltration post microfiltration after circulation certain time, collect permeate
Stand-by；
3) with the ratio of 1:1 (v/v), aseptic 0.1M PBS is added in the trapped fluid in head tank,
Resuspended, ON cycle pump circulation certain time, collect permeate, obtain the first washing filtrate stand-by；
4) with the ratio of 1:1 (v/v), aseptic 0.1M PBS is joined what step 3) was disposed
In trapped fluid in head tank, resuspended, ON cycle pump circulation certain time, collect permeate, obtain second
Washing filtrate is stand-by；
5) with the ratio of 1:1 (v/v), aseptic 0.1M PBS is joined what step 4) was disposed
In trapped fluid in head tank, resuspended, ON cycle pump circulation certain time, collect permeate, obtain the 3rd
Washing filtrate is stand-by；
6) by above-mentioned permeate, the first washing filtrate, the second washing filtrate, the 3rd washing filtrate mix homogeneously, obtain
Mixed liquor；
7) mixed liquor step 6) obtained injects the head tank that purification system is concentrated by ultrafiltration, ON cycle pump
Circulation certain time, carry out filtration treatment through 100KD or 300KD Hollow Fiber Ultrafiltration post, discard permeate,
It is collected in batch can remaining trapped fluid, is primary concentrating virus liquid；
It is 8) in primary concentrating virus liquid 0.1M PBS being injected in batch can with the ratio of 1:1 (v/v), resuspended,
ON cycle pump circulation certain time, discard permeate, be collected in batch can remain trapped fluid, be two grades dense
Contracting virus liquid；
It is 9) in secondary concentration virus liquid 0.1M PBS being injected in batch can with the ratio of 1:1 (v/v), resuspended,
ON cycle pump, circulates certain time, discards washing filtrate, is collected in batch can remaining trapped fluid, is three grades
Concentrating virus liquid；
10) in three grades of concentrating virus liquid 0.1M PBS being injected in batch can with the ratio of 1:1 (v/v), weight
Outstanding, ON cycle pump, circulates certain time, discards washing filtrate, be collected in batch can remaining trapped fluid, be
Viral concentration liquid finished product.
The viral concentration liquid finished product of above-mentioned preparation is stored in-20 DEG C.
Above-mentioned Latex agglutination test purification process, uses two step method of purification, and the first step passes through larger aperture
Clarification membrane filtration virus liquid, remove the cell debris in venom；Second step is clear by smaller aperture due
The filtered solution of the pureization filter membrane filter wash first step, reaches polysorbas20, the small molecular protein removing in virus liquid,
Impurity such as the small-molecule substance of products of cellular metabolism and realize the purposes such as concentrating virus.
Above-mentioned Latex agglutination test purification process, the oscillation treatment time described in step 1) is preferably 10min；
Ratio described in step 1) is preferably 5%；Step 2) described in the filter sizes of microfiltration be preferably 0.65 μm；
Ultrafiltration filter sizes described in step 7) is preferably 300KD；Step 2, in 3,4,5,7,8,9,10
The operating condition of described ON cycle pump circulation certain time is both preferably 400rpm and circulates 30min；Utilize
Viral concentration liquid finished product prepared by the method is for preparing vaccine；Virus clarification described above and concentration side
Method, step 2) in the volume of permeate can be adjusted as required, preferably permeate volume reaches former
The 90% of liquid；Cycles of concentration in step 7) can be adjusted according to actual needs, preferably concentration 10
More than Bei.
In technique scheme, the Latex agglutination test liquid described in step 1) refers to that the virus prepared is former
Liquid, not diluted processes；Step 1) adds polysorbas20 and plays emulsification, it is possible to be prevented effectively from virion
Son is assembled agglomerating, thus promotes subsequent filter efficiency.
Described microfiltration clarification system refers to that GE company manufactures FlexStand doughnut clarification system
System；Described micro-filtration membrane can manufacture selected from GE company, model is CFP-6-D-9A, CFP-4-E-9A's
Xample microfiltration post film；Described ultrafilter membrane can manufacture selected from GE company, model is UFP-300-C-9A,
The Xample ultrafiltration post film of UFP-100-C-9A.
The detection method of the viral concentration liquid finished product of above-mentioned preparation is as follows:
Virus sample in purified concentration technical process is carried out virus titer and protein concentration detects, to divide
The effectiveness of analysis concentration technology.Wherein the detection method of virus titer is:
TCID is measured with observation of cell pathological changes50Method the virus titer of each sample is detected, purification has
Effect criterion be: concentrate and purify and repeat filter wash washing filtrate inspection do not measure survival virus；The disease of concentrated solution
The poison response rate is more than 80%.
The assay method of protein content is:
With determination of protein concentration test kit, the albumen remaining quantity of concentrating virus liquid is measured, soluble protein
Less than the 10% of stock solution soluble protein, remaining quantity should show that technique is effective.
Above the summary of the invention of the present invention is described in detail.The purification process of the present invention comprehensively have employed micro-
Filter clarification method, is concentrated by ultrafiltration method of purification, repeats the series of process such as filter wash method, increases to greatest extent
The organic efficiency of JEV, reduces purification cost.The Latex agglutination test concentrated solution finished product prepared with the present invention
It is particularly suited for preparing vaccine, vaccine phase prepared by the Latex agglutination test concentrated solution finished product prepared with the present invention
For the Latex agglutination test vaccine of prior art, having a high purity, safety is high, and homogeneity is good, exempt from
The advantages such as epidemic disease is effective, meanwhile, present invention process is easy, cost is relatively low, before having prominent scale application
Scape.
Detailed description of the invention
In embodiment, instrument model is as follows:
FlexStand doughnut clarification system；
Xample microfiltration post: CFP-6-D-9A (0.65 μm), CFP-4-E-9A (0.45 μm)；
Xample ultrafiltration post: UFP-300-C-9A (300KD), UFP-100-C-9A (100KD)；
Above-mentioned instrument is purchased from GE company.
In embodiment, agents useful for same is as follows:
100L Latex agglutination test liquid, 108.5TCID50/ ml, is produced by our company；
BCA protein quantification test kit, is century purchased from health.
JEV antibody diagnosing reagent kit, Beijing Biological Product Inst. produces.
0.5M NaOH100L；
Aseptic 0.1M PBS100L；
Aseptic high purity water 100L；
Polysorbas20, analytical pure；
Sucrose gelatin protective agent.
Embodiment 1
1 operating process
1.1 pre-treatment
1.1.1 the assembling of system
Under aseptic condition, clarification system and concentration systems are assembled according to assembling requirement.In clarification system
System can be installed aseptic 0.65 μm, intact hollow fiber microfiltration membrane, concentration systems is installed aperture
Aseptic hollow fiber ultrafiltration membrane for 300KD.
1.1.2 system integrity detection
Pressure keeps the integrity of method detection purification system.
1.1.3 the process of system
Clean and sterilizing:
0.5M NaOH solution is filled the head tank of purification system, immersion treatment 20min, ON cycle pump
300rpm, carries out cleaning and sterilization treatment 30min of system.
Washing and flux detection:
After sterilizing terminates, drain intrasystem NaOH solution.Aseptic ultra-pure water is filled the head tank of system,
ON cycle pump, 300rpm circulates 30min, abandons the most intrasystem liquid, the most repeatedly wash, until being
In system, PH is about 7.0.
PBS process:
After washing terminates, abandon last to the greatest extent ultra-pure water.0.1M PBS solution is filled head tank, and unlatching follows
Ring pump 300rpm circulation flushing 20min.
The clarification of 1.2 viruses
1.2.1 the process of virus liquid
Polysorbas20 aseptic for 5L is added in 100L Latex agglutination test (JEV) virus liquid, at vibration
Reason 10min.
1.2.2 the clarification of virus liquid
After clarification system is disposed, abandon the most intrasystem PBS solution, by the virus liquid after oscillation treatment,
Injecting in the head tank of clarification system, ON cycle pump, 400rpm circulates 30min, in 0.65 μm
Hollow fiber microfiltration post is purified process, collects permeate 90L, retains trapped fluid 10L in head tank.
1.2.3 the filter wash of trapped fluid
Filter wash for the first time:
0.1M PBS aseptic for 10L is added in the trapped fluid in head tank, resuspended, ON cycle pump 400rpm
Circulation 30min, collects permeate 10L, is designated as washing filtrate 1.
Filter wash for the second time:
0.1M PBS aseptic for 10L is added in the trapped fluid in head tank, resuspended, ON cycle pump 400rpm
Circulation 30min, collects permeate 10L, is designated as washing filtrate 2.
Filter wash for the third time:
0.1M PBS aseptic for 10L is added in the trapped fluid in head tank, resuspended, ON cycle pump 400rpm
Circulation 30min, collects permeate 10L, is designated as washing filtrate 3.
1.2.4 the collection of virus liquid
By the permeate collected by above-mentioned clarification process and three washing filtrate mix homogeneously, obtain mixed liquor
120L。
1.2.5 primary concentration
After concentration systems is disposed, mixed liquor is injected the head tank of concentration systems, ON cycle pump,
400rpm circulates 30min, is purified process through 300KD Hollow Fiber Ultrafiltration post, discards permeate, enter
Batch can remains trapped fluid 10L, is designated as primary concentrating virus liquid.
1.2.6 the filter wash of concentrated solution
Filter wash for the first time:
In the primary concentrated solution that 10L0.1M PBS is injected in batch can, resuspended, ON cycle pump, 400rpm
Circulation 30min, discards permeate 10L, remains trapped fluid 10L, be designated as secondary concentration virus liquid in head tank.
Filter wash for the second time:
The secondary concentration liquid that 10L0.1M PBS is injected in batch can, resuspended, ON cycle pump, 400rpm
Circulation 30min, discards washing filtrate 10L, remains trapped fluid 10L, be designated as three grades of concentrating virus liquid in head tank.
Filter wash for the third time:
Three grades of concentrated solutions that 10L0.1M PBS is injected in batch can, resuspended, ON cycle pump, 400rpm
Circulation 30min, discards washing filtrate 10L, remains trapped fluid 10L, be viral concentration liquid finished product in head tank.
2.2.7 the collection of virus liquid
The viral concentration liquid finished product obtained after above-mentioned process is collected, is stored in-20 DEG C.
2 validation checkings
Virus sample in purified concentration technical process is carried out virus titer and protein concentration detects, to divide
The effectiveness of analysis concentration technology.
The detection of 2.1 virus titers
TCID is measured with observation of cell pathological changes50Method the virus titer of each sample is detected, specifically walk
Rapid as follows:
2.1.1 the bed board of cell and cultivation
EDTA-pancreatin with 0.05% is by well-grown detection young hamster kidney passage cell (BHK-21)
Digest, resuspended for 1 × 10 with cell growth medium5/ ml is also inoculated in 96 porocyte culture plates, 100 μ l/ holes,
It is placed in 37 DEG C, 5%CO2Cell culture incubator is cultivated 24h.
2.1.2 the gradient dilution of virus
By the venom sample of each collection, carry out 10 gradients of 10 times of doubling dilutions with 0.1M PBS
(10-1～10-10).
2.1.3 viral inoculation and cultivation
The detection cell described in step (1) will be inoculated in by each gradient virus liquid after dilution, 100 μ l/ holes, each
Gradient 6 repetition, arranges the cell controls not connecing virus liquid simultaneously, 37 DEG C, 5%CO2In cell culture incubator
Maintain and cultivate 5～7 days.
2.1.4 add up and calculate virus titer
There is cytopathic hole count in statistics, with the Reed-Muench method titre to Latex agglutination test
(TCID50) calculate, as a result, it was confirmed that the titre of the virus liquid after Nong Suoing is apparently higher than the virus before concentration
Liquid, the washing filtrate of enriching stage and the most acellular pathological changes of cell controls group, concentrate restrovirus titre and reach
109.0TCID50/ ml, the washing filtrate of enriching stage is without titer, and viral recovery is close to 100%.
The detection of 2.2 soluble proteins
Taking each sample to be measured with BCA protein concentration test kit, being computed soluble protein survival rate is
9.5%。
Prepared by 3 vaccines
The viral concentration liquid prepared using above-mentioned technique, as raw material, prepares vaccine further, and its processing step is as follows:
3.1 Seedling is joined in dilution
JEV venom after purified concentration is diluted with aseptic 0.1M PBS so that it is virus titer reaches
108.5TCID50/ ml, by JEV virus liquid (10 before purification8.5TCID50/ ml) with JEV virus liquid after purification
(108.5TCID50/ ml) it is separately added into suitable sucrose gelatin protective agent, carry out joining Seedling；JEV before purification is sick
Vaccine prepared by venom and JEV virus liquid after purification is designated as respectively: JEV inactivated vaccine 1 and JEV inactivates
Vaccine 2.
3.2 lyophilizing
Two parts are joined Seedling liquid, in mix homogeneously, and subpackage respectively to cillin bottle, every bottle of subpackage 2.3ml.Use
(goods are in less than-30 DEG C pre-freeze 3h for identical lyophilizing program；Temperature programmed control :-18 DEG C of 30min；-8℃30min；
-8℃15h；0℃30min；0℃5h；10℃2h；25℃30min；25 DEG C of 4h) carry out the lyophilizing of vaccine.
4 vaccine safety inspections
The JEV live vaccine 1(prepared with above-mentioned technique is before purification), JEV live vaccine 2(is after purification), aseptic
0.1M PBS solution is as experiment material；With the healthy susceptible piglets (JEV HI negative antibody) 12 of 4～8 ages in days
Head and 10～12g SPF mices 30 are only used as laboratory animal.Concrete operation step is as follows:
4.1 animal packets
12 piglets are randomly divided into 3 groups, often group 4.
4.2 vaccine immunity
4.2.1 piglets safety examination two kinds of vaccine 2ml(of intramuscular injection respectively are containing 10 parts) in experiment breast
Pig, matched group injection 2ml PBS, often organizes each injection 4.Observe 21.
4.2.1 mice safety examination two kinds of vaccine 0.1ml(of subcutaneous injection respectively are containing 0.5 part) use in experiment
Mice, matched group injection 0.1ml PBS, simultaneously on the right side of empty thorn in brain, often organize each 4, observe 14.
4.3 experimental result
Latter 21 days of immunity, each group piglets is all without the locally or systemically untoward reaction occurred because of vaccination；Exempt from
After epidemic disease 14 days, each group mice is the most strong to live.
5 vaccine potency inspections
The detection of vaccine virus titre after 5.1 lyophilizing
JEV live vaccine 1 and JEV live vaccine 2 after lyophilizing is dissolved with 1ml PBS respectively and carries out virus
The detection of titre, method is with " 3.1 ".After result lyophilizing, the titre of JEV live vaccine 1 is 107.67TCID50/ ml,
JEV live vaccine 2 is 108.0TCID50/ml.I.e. after lyophilizing, JEV live vaccine 2(is after purification) lyophilizing loss
Rate is less than JEV live vaccine 1(before purification).
5.2 replacement gilt immunity tests
With JEV live vaccine 1, JEV live vaccine 2, aseptic 0.1M PBS solution, as experiment reagent, utilizes
JEV antibody diagnosing reagent kit measures JEV HI antibody；Take 70～80kg healthy susceptible replacement gilt (JEV
HI negative antibody) 12 as laboratory animal.Specific experiment step is as follows:
5.2.1 experimental animal packet
12 sows are randomly divided into 3 groups, often group 4.
5.2.2 vaccine immunity
Musculi colli two kinds of vaccines of injection and PBS2ml are in test replacement gilt respectively, often organize each injection 4
Head.
5.2.3 blood sampling and antibody test
Take a blood sample weekly once before immunity and after immunity, totally 6 times, separate serum, use JEV antibody diagnosis
Test kit detects.
5.2.4 experimental result
Test result indicate that, by JEV live vaccine 2(after purification) immunity second group of sow antibody horizontal produce
Raw relatively morning is also higher than JEV live vaccine 1(before purification).
In sum, the Latex agglutination test utilizing purification of the present invention to obtain concentrates and purifies liquid finished product and is applicable to system
Standby Latex agglutination test vaccine, its safety is good, immune efficacy is notable.
Embodiment 2
1) aseptic polysorbas20 is added in Latex agglutination test (JEV) virus liquid in the ratio of 10% (v/v),
Oscillation treatment 10min；
2) virus liquid after step 1) oscillation treatment is injected in the head tank of microfiltration clarification system, open
Open circulating pump, through 0.45 μm doughnut microfiltration post microfiltration after circulation certain time, collect permeate stand-by；
3) with the ratio of 1:1 (v/v), aseptic 0.1M PBS is added in the trapped fluid in head tank,
Resuspended, ON cycle pump circulation certain time, collect permeate, obtain the first washing filtrate；
4) with the ratio of 1:1 (v/v), aseptic 0.1M PBS is joined what step 3) was disposed
In trapped fluid in head tank, resuspended, ON cycle pump circulation certain time, collect permeate, obtain second
Washing filtrate is stand-by；
5) with the ratio of 1:1 (v/v), aseptic 0.1M PBS is joined what step 4) was disposed
In trapped fluid in head tank, resuspended, ON cycle pump circulation certain time, collect permeate, obtain the 3rd
Washing filtrate is stand-by.
6) by above-mentioned permeate, the first washing filtrate, the second washing filtrate, the 3rd washing filtrate mix homogeneously, obtain
Mixed liquor；
7) mixed liquor step 6) obtained injects the head tank that purification system is concentrated by ultrafiltration, ON cycle pump
Circulation certain time, carry out filtration treatment through 100KD Hollow Fiber Ultrafiltration post, discard permeate, be collected into
Batch can remains trapped fluid, is primary concentrating virus liquid；
It is 8) in primary concentrating virus liquid 0.1M PBS being injected in batch can with the ratio of 1:1 (v/v), resuspended,
ON cycle pump circulation certain time, discard permeate, be collected in batch can remain trapped fluid, be two grades dense
Contracting virus liquid；
It is 9) in secondary concentration virus liquid 0.1M PBS being injected in batch can with the ratio of 1:1 (v/v), resuspended,
ON cycle pump, circulates certain time, discards washing filtrate, is collected in batch can remaining trapped fluid, is three grades
Concentrating virus liquid；
10) in three grades of concentrating virus liquid 0.1M PBS being injected in batch can with the ratio of 1:1 (v/v), weight
Outstanding, ON cycle pump, circulates certain time, discards washing filtrate, be collected in batch can remaining trapped fluid, be
Viral concentration liquid finished product.
Embodiment 3
1) aseptic polysorbas20 is added in Latex agglutination test (JEV) virus liquid in the ratio of 0.5% (v/v),
Oscillation treatment 10min；
2) virus liquid after step 1) oscillation treatment is injected in the head tank of microfiltration clarification system, open
Open circulating pump and circulate 30min with the condition of 400rpm, and after through 0.65 μm doughnut microfiltration post microfiltration, receive
Collection permeate is stand-by；
3) with the ratio of 1:1 (v/v), aseptic 0.1M PBS is added in the trapped fluid in head tank,
Resuspended, ON cycle pump circulates 30min with the condition of 400rpm, collects permeate, obtains the first washing filtrate
Stand-by；
4) with the ratio of 1:1 (v/v), aseptic 0.1M PBS is joined what step 3) was disposed
In trapped fluid in head tank, resuspended, ON cycle pump circulates 30min with the condition of 400rpm, collects thoroughly
Cross liquid, obtain that the second washing filtrate is stand-by to be deposited；
5) with the ratio of 1:1 (v/v), aseptic 0.1M PBS is joined what step 4) was disposed
In trapped fluid in head tank, resuspended, ON cycle pump circulates 30min with the condition of 400rpm, collects thoroughly
Cross liquid, obtain the 3rd washing filtrate stand-by.
6) by above-mentioned permeate, the first washing filtrate, the second washing filtrate, the 3rd washing filtrate mix homogeneously, obtain
Mixed liquor；
7) mixed liquor that step 6) obtained injects the head tank of ultrafiltration concentration system, ON cycle pump with
The condition circulation 30min of 400rpm, carries out hyperfiltration treatment through 300KD Hollow Fiber Ultrafiltration post, discards and pass through
Liquid, is collected in batch can remaining trapped fluid, is primary concentrating virus liquid；
It is 8) in primary concentrating virus liquid 0.1M PBS being injected in batch can with the ratio of 1:1 (v/v), resuspended,
ON cycle pump circulates 30min with the condition of 400rpm, discards permeate, is collected in batch can residue and retains
Liquid, is secondary concentration virus liquid；
It is 9) in secondary concentration virus liquid 0.1M PBS being injected in batch can with the ratio of 1:1 (v/v), resuspended,
ON cycle pump circulates 30min with the condition of 400rpm, discards washing filtrate, is collected in batch can residue and retains
Liquid, is three grades of concentrating virus liquid；
10) in three grades of concentrating virus liquid 0.1M PBS being injected in batch can with the ratio of 1:1 (v/v), weight
Outstanding, ON cycle pump circulates 30min with the condition of 400rpm, discards washing filtrate, is collected in batch can residue
Trapped fluid, is viral concentration liquid finished product.
Above the embodiment of the present invention is described in detail, but described content has been only the preferable enforcement of the present invention
Example, not in order to limit the present invention.All made in the application range of the present invention any amendment, equivalent
With improvement etc., should be included within the scope of the present invention.
Claims (1)
1. a Latex agglutination test purification process, it is characterised in that use microfiltration clarification system and surpass
Filter concentrating and purifying system realizes the purification of virus liquid, and comprises the following steps:
1.1 pre-treatment
1.1.1 the assembling of system
Under aseptic condition, clarification system and concentration systems are assembled according to assembling requirement；In clarification system
System is installed aseptic 0.65 μm, intact hollow fiber microfiltration membrane, concentration systems is installed aperture
Aseptic hollow fiber ultrafiltration membrane for 300KD；
1.1.2 system integrity detection
Pressure keeps the integrity of method detection purification system；
1.1.3 the process of system
Clean and sterilizing:
0.5M NaOH solution is filled the head tank of purification system, immersion treatment 20min, ON cycle pump
300rpm, carries out cleaning and sterilization treatment 30min of system；
Washing and flux detection:
After sterilizing terminates, drain intrasystem NaOH solution；Aseptic ultra-pure water is filled the head tank of system,
ON cycle pump, 300rpm circulates 30min, abandons the most intrasystem liquid, the most repeatedly wash, until being
In system, PH is 7.0；
PBS process:
After washing terminates, abandon last to the greatest extent ultra-pure water；0.1M PBS solution is filled head tank, and unlatching follows
Ring pump 300rpm circulation flushing 20min；
The clarification of 1.2 viruses
1.2.1 the process of virus liquid
Polysorbas20 aseptic for 5L is added in 100L Latex agglutination test (JEV) virus liquid, at vibration
Reason 10min；
1.2.2 the clarification of virus liquid
After clarification system is disposed, abandon the most intrasystem PBS solution, by the virus liquid after oscillation treatment,
Injecting in the head tank of clarification system, ON cycle pump, 400rpm circulates 30min, in 0.65 μm
Hollow fiber microfiltration post is purified process, collects permeate 90L, retains trapped fluid 10L in head tank；
1.2.3 the filter wash of trapped fluid
Filter wash for the first time:
0.1M PBS aseptic for 10L is added in the trapped fluid in head tank, resuspended, ON cycle pump 400rpm
Circulation 30min, collects permeate 10L, is designated as washing filtrate 1；
Filter wash for the second time:
0.1M PBS aseptic for 10L is added in the trapped fluid in head tank, resuspended, ON cycle pump 400rpm
Circulation 30min, collects permeate 10L, is designated as washing filtrate 2；
Filter wash for the third time:
0.1M PBS aseptic for 10L is added in the trapped fluid in head tank, resuspended, ON cycle pump 400rpm
Circulation 30min, collects permeate 10L, is designated as washing filtrate 3；
1.2.4 the collection of virus liquid
By the permeate collected by above-mentioned clarification process and three washing filtrate mix homogeneously, obtain mixed liquor
120L；
1.2.5 primary concentration
After concentration systems is disposed, mixed liquor is injected the head tank of concentration systems, ON cycle pump,
400rpm circulates 30min, is purified process through 300KD Hollow Fiber Ultrafiltration post, discards permeate, enter
Batch can remains trapped fluid 10L, is designated as primary concentrating virus liquid；
1.2.6 the filter wash of concentrated solution
Filter wash for the first time:
In the primary concentrated solution that 10L 0.1M PBS is injected in batch can, resuspended, ON cycle pump, 400rpm
Circulation 30min, discards permeate 10L, remains trapped fluid 10L, be designated as secondary concentration virus liquid in head tank；
Filter wash for the second time:
The secondary concentration liquid that 10L 0.1M PBS is injected in batch can, resuspended, ON cycle pump, 400rpm
Circulation 30min, discards washing filtrate 10L, remains trapped fluid 10L, be designated as three grades of concentrating virus liquid in head tank；
Filter wash for the third time:
Three grades of concentrated solutions that 10L 0.1M PBS is injected in batch can, resuspended, ON cycle pump, 400rpm
Circulation 30min, discards washing filtrate 10L, remains trapped fluid 10L, be viral concentration liquid finished product in head tank；
2.2.7 the collection of virus liquid
The viral concentration liquid finished product obtained after above-mentioned process is collected, is stored in-20 DEG C.
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