CN107748255A - Gosling plague antibody test standard substance and preparation method thereof - Google Patents
Gosling plague antibody test standard substance and preparation method thereof Download PDFInfo
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- CN107748255A CN107748255A CN201710738053.7A CN201710738053A CN107748255A CN 107748255 A CN107748255 A CN 107748255A CN 201710738053 A CN201710738053 A CN 201710738053A CN 107748255 A CN107748255 A CN 107748255A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/465—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from birds
Abstract
The invention discloses a kind of gosling plague antibody test standard substance and preparation method thereof.Gosling plague antibody test standard substance of the present invention includes:Gosling plague agp antigen, gosling plague positive serum and gosling plague negative serum.Wherein, the preparation of the gosling plague agp antigen includes:Goose parvovirus strain is inoculated with susceptible goose embryo, goose blastochyle is harvested, obtains antigen liquid；Freeze drying protectant is added into antigen liquid, is freeze-dried and produces.The preparation of the gosling plague positive serum includes:Goose parvovirus strain is inoculated with susceptible goose embryo, goose blastochyle is harvested, obtains antigen liquid；Gosling plague immunogene will be prepared after the concentrated inactivation of antigen liquid, the susceptible goose of immune health, blood sampling, serum is separated, is freeze-dried and produces.The preparation of the gosling plague negative serum includes:Goose blood sampling is negative into gosling plague antibody, serum is separated, is freeze-dried and produces.Gosling plague antibody test standard substance of the present invention it is specific good, definite value accuracy is high, has application prospect in gosling plague prevention and control.
Description
Technical field
The present invention relates to gosling plague antibody test standard substance, the system of the gosling plague antibody test standard substance is further related to
Preparation Method, belong to the preparation field of gosling plague antibody test standard substance.
Background technology
Gosling plague is that the young goose caused by goose parvovirus (Goose Parvovirus, GPV) is acute or subacute sepsis
Sexually transmitted disease.The disease is that Chinese scholar Fang Dingyi had found in Jiangsu Yangzhou first in 1956, since the sixties, in Europe, Asia
The country such as continent reports the sick generation and prevalence in succession.1974, the disease was formally named as by international disease of poultry meeting (WPSA)
DerzsysShi diseases.The disease mainly encroaches on out the young goose of 4~20 ages in days after shell, also infects duckling, and spread speed is fast, the incidence of disease and
The death rate is up to 90%~100%, particularly has prevalence in some Chinese provinces, municipalities and autonomous regions in recent years, provisions goose industry causes
Seriously endanger, great economic loss is brought, by the extensive concern of China and outside China animal and veterinary worker.
However, the quality requirement of gosling plague antibody test standard substance is high, preparation method and inspection, scaling method are strict,
It is not reported so far about preparation procedure and scaling method, therefore the difficulty such as Viral diagnosis, the examination and test of products, scientific research increases,
Experimental result is easily influenceed by the experience of personnel, ability and Experimental Establishment, environment and larger error be present, is seriously constrained small
The prevention and control of goose pest.Therefore, need badly using standard substance as objective, just scale, for control of product quality, scientific research
The ability between reference and laboratory with laboratory diagnosis compares.
Herbal polysaccharide, particularly help class herbal polysaccharide have the function that enhancing body's immunity, and safe and nontoxic,
It is good BRM, is expected to be developed into new vaccine adjuvant.Experiment confirms that astragalus polyose can significantly improve machine
Body immunity function, there is immunopotentiation to a variety of vaccines.At present, gosling plague bacterin co-immunization is coordinated using astragalus polyose
Related content, have not been reported.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of gosling plague antibody test standard substance and preparation method thereof,
Gosling plague antibody test standard substance specificity prepared by the present invention is good, applied widely, and definite value accuracy is high.
In order to solve the above technical problems, the technical solution used in the present invention is：
The present invention discloses a kind of gosling plague antibody test standard substance first, including:Gosling plague agp antigen, gosling plague
Positive serum and gosling plague negative serum.
The present invention further discloses the preparation method of the gosling plague antibody test standard substance.
Wherein, the preparation of the gosling plague agp antigen includes:(1) by goose parvovirus (Goose Parvovirus,
GPV) strain is inoculated with susceptible goose embryo, harvests goose blastochyle, obtains antigen liquid；(2) freeze drying protectant is added into antigen liquid, is mixed,
Freeze-drying, is produced.
Step (1) the goose parvovirus strain is preferably goose parvovirus H strains, and its microbial preservation numbering is:CGMCC
No.6851。
Goose parvovirus H strains of the present invention are separated by biovaccine company of Harbin Pharmaceutical Group, and through negative staining electron microscope Appearance View
Examine, goose embryo infection neutralization test, viral genome PCR detections and nucleotide sequencing etc., be defined as goose parvovirus, name
For goose parvovirus H strains (GPV-H), (China of China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in
Patent of invention ZL201210543550.9).
The viral level of step (1) described antigen liquid is:Per viral level >=10 of 0.2ml antigen liquids5.0ELD50(goose embryo
Median lethal dose)；Step (1) the susceptible goose embryo is preferably the susceptible goose embryo of 12 ages in days；The goose blastochyle is small to be incubated 72-168
When interior dead goose embryo goose blastochyle；The temperature of the incubation is 37 DEG C.Step (2) is according to volume basis, antigen liquid:It is lyophilized to protect
Protect agent=1-3:1-3, preferably 1:1.The freeze drying protectant is made up of sucrose, skimmed milk and water；Preferably, based on g/ml,
Final concentration of the 2.5% of sucrose in the freeze drying protectant, final concentration of the 2.5% of skimmed milk, surplus is water.
In gosling plague antibody test standard substance of the present invention, antibody titer >=1 of the gosling plague positive serum:
32 (through AGP test measurings).
The preparation of gosling plague positive serum of the present invention includes:(1) goose parvovirus strain is inoculated with susceptible goose embryo, received
Goose blastochyle is obtained, obtains antigen liquid；(2) antigen liquid is concentrated, inactivated, obtained inactivation antigen prepares gosling plague immunogene；(3) will
The susceptible goose of gosling plague immunogen immune health, blood sampling, serum is separated, freeze-drying, is produced.
In the preparation method of the gosling plague positive serum, step (1) the goose parvovirus strain is preferably that goose is tiny
Viral H strains, its microbial preservation numbering are:CGMCC No.6851；The goose blastochyle is goose dead in incubation 72-168 hours
The goose blastochyle of embryo (temperature of the incubation is 37 DEG C)；The viral level of the antigen liquid is:Virus per 0.2ml antigen liquids contains
Amount >=105.0ELD50；The susceptible goose embryo is preferably the susceptible goose embryo of 12 ages in days.Goose blastochyle of the present invention includes allantoic fluid and sheep
Water.
Step (2) concentration is that antigen liquid is carried out into 10 times of concentrations；Preferably, the concentration includes：By antigen liquid from
The heart, supernatant is taken, the milipore filter bag for being 50kDa with molecular cut off carries out 10 times of concentrations；The inactivation includes:By volume
Meter, final concentration of 0.2% formalin (percent by volume) is added into the antigen after concentration, is mixed, and 37 DEG C inactivate 48 hours
(hour period 6-8 shakes 1 time).
The preparation of step (2) the gosling plague immunogene includes:(a) inactivation antigen is well mixed with Tween-80, obtained
Aqueous phase；(b) injection white oil, aluminum stearate are well mixed with Si Ben -80, sterilize, obtain oil phase；(c) by aqueous phase and oil phase
Mixing, emulsification, is produced.Wherein, step (a) is counted by volume, inactivation antigen:Tween-80=96:4；Step (b) is by volume
Meter, injection white oil:Aluminum stearate:Si Ben -80=94:1.5:6；Step (b) is preferably, first by injection white oil and stearic acid
Aluminium mixes, until it is fully transparent, add Si Ben -80；Step (c) is counted by volume, aqueous phase:Oil phase=1:1.5.
Step (3) the immune program includes:Fundamental immunity, carry out booster immunization within 14 days after fundamental immunity, strengthen exempting from
Carry out reinforced immunological within 14 days after epidemic disease.Wherein, the dosage of inoculation of the fundamental immunity is:Gosling plague immunogene 1.0-2.0ml and Huang
Astragalus polysaccharides parenteral solution 1.0ml；The dosage of inoculation of the booster immunization is:Gosling plague immunogene 1.0-3.0ml and astragalus polyose note
Penetrate liquid 1.0ml；The dosage of inoculation of the reinforced immunological is:Gosling plague immunogene 1.0-3.0ml and Radix Astragali polysaccharide injection
1.0ml；Preferably, the dosage of inoculation of the fundamental immunity is:Gosling plague immunogene 1.0ml and Radix Astragali polysaccharide injection 1.0ml；
The dosage of inoculation of the booster immunization is:Gosling plague immunogene 2.0ml and Radix Astragali polysaccharide injection 1.0ml；The reinforced immunological
Dosage of inoculation be:Gosling plague immunogene 3.0ml and Radix Astragali polysaccharide injection 1.0ml.The immune route of inoculation is muscle
Injection, preferably chest muscle multi-point injection.Step (3) is described to take a blood sample to be taken a blood sample within 14th after reinforced immunological.
The content of astragalus polyose is 20mg/ml in Radix Astragali polysaccharide injection of the present invention；The astragalus polyose uses water
Propose alcohol deposition method extraction.
The present invention shows the dosage of inoculation optimum results of gosling plague immunogene in gosling plague immune programme for children, fundamental immunity,
The inoculum concentration of booster immunization and reinforced immunological is respectively 1.0,2.0 and 3.0ml, either 2.0,2.0 and 3.0ml or 2.0,3.0
And 3.0ml, above-mentioned three kinds of immune programme for children reinforced immunologicals antibody fine jade on the 14th expand antibody titer geometrical mean and connect apparently higher than other
Kind dosage.The present invention considers the dosage of immunogene and the injury to animal body, determines that the optimal dosage of inoculation of immunogene is：
The dosage of inoculation of fundamental immunity is gosling plague immunogene 1.0ml；Booster immunization is carried out within 14 days after fundamental immunity, dosage of inoculation is small
Goose pest immunogene 2.0ml；Carry out reinforced immunological within 14 days after booster immunization, dosage of inoculation is gosling plague immunogene 3.0ml.
The present invention is further on the basis of the optimal dosage of inoculation of immunogene, in fundamental immunity, booster immunization and reinforced immunological
Increase Radix Astragali polysaccharide injection 1.0ml respectively while middle inoculation gosling plague immunogene.From the point of view of antibody produces potency, add the Radix Astragali
Polysaccharide group antibody titer geometrical mean on the 14th after reinforced immunological reaches 1:119.43, than not adding astragalus polyose group antibody titer
Geometrical mean improves nearly 2 times.Illustrate, astragalus polyose can improve the antibody titer of goose only after gosling plague immunogen immune.Therefore,
Present invention determine that in immune programme for children, the dosage of inoculation of fundamental immunity is:Gosling plague immunogene 1.0ml and Radix Astragali polysaccharide injection
1.0ml；The dosage of inoculation of booster immunization is:Gosling plague immunogene 2.0ml and Radix Astragali polysaccharide injection 1.0ml；Reinforced immunological
Dosage of inoculation is:Gosling plague immunogene 3.0ml and Radix Astragali polysaccharide injection 1.0ml.
In gosling plague antibody test standard substance of the present invention, the preparation of the gosling plague negative serum includes:It is right
What gosling plague antibody was negative takes a blood sample into goose, separates serum, freeze-drying, produces.
Separation serum of the present invention includes:By the blood of collection, put 37 DEG C and act on 1 hour, 4 DEG C act on 1 hour, nothing
Bacterium separates serum, produces.
The chilled vacuum drying of gosling plague positive serum and negative serum of the present invention, it can be not required to add frozen-dried protective
Agent.
The present invention has carried out physical behavior inspection, steriling test, special to described gosling plague antibody test standard substance
Property examine, potency examine and residual moisture measure etc..
Infections chicken cloacal bursa virus, Newcastle Disease are not contained in gosling plague agp antigen standard substance of the present invention
Poison, marek's disease virus, fowl influenza A, aviadenovirus, Avianreovirus, bird pox virus, avian infectious bronchus
Scorching virus, avian infectious laryngotracheitis virus, reticuloendothiliosis virus, fowl cell leukemia virus and fowl brain ridge
Marrow inflammation virus etc..Infections chicken cloacal bursa, ewcastle disease, Marek's are not contained in the gosling plague positive serum standard substance
Disease, fowl A types influenza, fowl exhale the associated antibodies such as the lonely disease of intestines and chicken pox.Do not contained in the gosling plague negative serum standard substance small
Goose pest, infections chicken cloacal bursa, ewcastle disease, Marek's disease, fowl A types influenza, fowl exhale the associated antibodies such as the lonely disease of intestines and chicken pox.
Gosling plague antibody test standard substance of the present invention can be applied to prepare detection gosling plague (DerzsysShi diseases)
Reagent, effective prevention and control for gosling plague have important value.
Technical solution of the present invention compared with prior art, has the advantages that：
It is the preparation method science of gosling plague antibody test standard substance of the present invention, rigorous, perfect.Present invention application is yellow
Astragalus polysaccharides coordinate gosling plague immunogene co-immunization, hence it is evident that improve the antibody titer of goose only after gosling plague immunogen immune.This
The described gosling plague antibody test standard substance of invention, specificity is good, applied widely, and definite value accuracy is high, in gosling plague
There is important application prospect in detection or prevention and control.
Brief description of the drawings
Fig. 1 is that gosling plague agp antigen prepares and examined process chart；
Fig. 2 is that gosling plague positive serum prepares and examined process chart；
Fig. 3 is that gosling plague negative serum prepares and examined process chart.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.It should be understood that the embodiment is only exemplary, any restrictions are not formed to the scope of the present invention.This area
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or replaced, but these modifications or substitutions each fall within protection scope of the present invention.
1st, virus stain
Goose parvovirus H strains (GPV-H), separated by biovaccine company of Harbin Pharmaceutical Group, be preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center, deposit number are:CGMCC No.6851.
The gosling plague agp antigen of embodiment 1 is prepared and examined
1st, the preparation of seed culture of viruses
By goose parvovirus H strains, (preserving number is:CGMCC No.6851) with physiological saline make 1000 times dilution, allantoic cavity
The susceptible goose embryo of 12 ages in days is inoculated with, per embryo 0.2ml.37 DEG C of incubations are put, goose embryo dead in 72~168 hours is taken out, collects goose embryo
Liquid, loaded in sterile chamber, while draw part goose blastochyle and do steriling test and viral level measure, qualified goose embryo will be examined
Liquid is placed in -20 DEG C of preservations.
2nd, preparing and packaging
Goose blastochyle proportionally 1:It is (described lyophilized based on g/ml that 1 (volume ratio), 5% sucrose defatted milk of addition makees protective agent
Final concentration of the 2.5% of sucrose in protective agent, final concentration of the 2.5% of skimmed milk, surplus is water), quantitative separating is in glass tube vial
It is interior.
3rd, freeze
Vacuum freezedrying is carried out, by 2000 editions《Code》Annex progress of page 437.Put -20 DEG C of preservations.
4th, seed culture of viruses is examined
4.1 character
Faint yellow Sponge Porosity agglomerate, easily depart from bottle wall, dissolved rapidly after adding dilution.
It is 4.2 pure
By existing《Chinese veterinary pharmacopoeia》Annex is tested, and should be polluted without bacterium, mould, mycoplasma and exogenous virus.
4.3 viral level
Seed culture of viruses physiological saline work is serially diluted for 10 times, takes 10-4、10-5、10-6、10-74 dilution factors, each dilution
Degree is inoculated with 12 5 pieces of age in days goose embryos through allantoic cavity, per embryo 0.2ml, puts dead goose embryo before 37 DEG C of incubations are observed 168 hours, 72 hours
Disregard, record 72~168 hours goose embryo death conditions, calculate ELD50, >=10 are answered per 0.2ml viral levels5.0ELD50。
It is 4.4 specific
It is 200ELD that seed culture of viruses is diluted into viral level50/ 0.2ml, mixed with equivalent anti gosling plague virus specific serum,
Put in 37 DEG C of water-baths and after 1 hour, allantoic cavity inoculation susceptible 5 pieces of the goose embryo of 12 ages in days, per embryo 0.2ml；Set virus control group 5 simultaneously
Piece, the virus liquid and mixed liquor of normal saline 0.2ml handled per embryonic breeding kind with condition, 37 DEG C are incubated observation 168 hours, 72 hours
Preceding dead goose embryo is disregarded, and records 72~168 hours goose embryo death conditions.Neutralization group goose embryo should all be good for work, control group at least 4
Piece goose embryo is dead, and seed culture of viruses does chicken red blood cell agglutination test (HA) and should be negative.
4.5 residual moistures determine
By existing《Chinese veterinary pharmacopoeia》Annex is measured, and should meet regulation.
4.6 vacuums determine
By existing《Chinese veterinary pharmacopoeia》Annex is measured, and should meet regulation.
Gosling plague agp antigen prepares and examines technological process to see Fig. 1.
The optimization of the gosling plague immune programme for children of embodiment 2
1st, immunogenic dose optimizes
1.1 experiment packets
The susceptible goose 60 of 40 ages in days health is only divided into 6 groups, every group 10.Separately set normal healthy controls 10, isolated rearing simultaneously.
1.2 immunity inoculation
By gosling plague immunogene (preparation method is with embodiment 3), it is inoculated with according to the immune programme for children of table 1.Control group injects physiology
Salt solution, isolated rearing.
The gosling plague immune programme for children of table 1
1.3 antibody titers determine
After the susceptible goose of above-mentioned 6 groups of immune programme for children inoculation health, before fundamental immunity, before booster immunization, strengthen
With 14 days after reinforced immunological 4 time points collection susceptible goose blood 2.0ml of health before immune, put 37 DEG C and act on 1 hour, 4 DEG C of effects 1
Hour, sterile separation serum determines the antibody titer of the susceptible goose of every health for agar gel diffusion test, counted as measuring samples
Calculate the geometrical mean of AGP antibody titers.
The geometrical mean of 1.4 different time points antibody titers and analysis
Different time points antibody titer geometrical mean result of calculation is shown in Table 2.As can be seen from Table 2, reinforced immunological
Antibody fine jade on the 14th expands antibody titer geometrical mean and is ordered as the 6th group successively from high to low>3rd group>5th group>4th group>2nd group
>1st group, wherein the 3rd group, the 5th group and the 6th group antibody titer difference is not notable, it is contemplated that the dosage of immunogene and to animal
The injury of body, it is the optimal dosage of inoculation of immunogene to determine the 3rd group of immune programme for children, is shown in Table 3.
The different time points antibody titer geometrical mean result of table 2
The optimal dosage of inoculation of the immunogene of table 3
2nd, the application of astragalus polyose
2.1 experiment packets
The susceptible goose 20 of 40 ages in days health is only divided into 2 groups, every group 10.Separately set normal healthy controls 10, isolated rearing simultaneously.
2.2 immunity inoculation
By gosling plague immunogene (preparation method is with embodiment 3) and Radix Astragali polysaccharide injection (content 20mg/ml) according to
The immune programme for children of table 4 is inoculated with.Control group injecting normal saline, isolated rearing.
The immunization program of table 4
2.3 antibody titers determine
After the susceptible goose of above-mentioned 2 groups of immune programme for children inoculation health, before fundamental immunity, before booster immunization, strengthen
With 14 days after reinforced immunological 4 time points collection susceptible goose blood 2.0ml of health before immune, put 37 DEG C and act on 1 hour, 4 DEG C of effects 1
Hour, sterile separation serum determines the antibody titer of the susceptible goose of every health for agar gel diffusion test, counted as measuring samples
Calculate the geometrical mean of AGP antibody titers.
The geometrical mean of 2.4 different time points antibody titers and analysis
Different time points antibody titer geometrical mean result of calculation is shown in Table 5.From the point of view of antibody produces potency, add the Radix Astragali more
Sugared group after reinforced immunological antibody titer geometrical mean on the 14th can reach 1:119.43, than not adding astragalus polyose group antibody titer
Geometrical mean improves nearly 2 times, determines that astragalus polyose can improve the antibody titer of goose only after gosling plague immunogen immune.
The different time points antibody titer geometrical mean result of table 5
The gosling plague positive serum of embodiment 3 is prepared and examined
1st, prepared by immunogene
1.1 inoculation
Goose parvovirus H strain production seeds culture of viruses are made into 1000 times of dilutions with physiological saline, it is susceptible that allantoic cavity is inoculated with 12 ages in days
Goose embryo, per embryo 0.2ml.Put 37 DEG C of incubations, it is not necessary to egg-turning.
1.2 are incubated and observe
After inoculation, goose embryo dead before 72 hours is discarded.After 72 hours, every 4~8 hours photograph eggs 1 time, 72~
Dead goose embryo takes out at any time in 168 hours, and air chamber stands on 2~8 DEG C of coolings upwards.
1.3 harvest
The goose embryo for cooling down 6~12 hours is taken out, with iodine tincture disinfection air chamber position, goose blastochyle is then collected with sterile working
(allantoic fluid and amniotic fluid), while harvest, the lesion of idiosome should be checked one by one, will without lesion idiosome, corrupt blastochyle it is muddy and
Any pollution suspicious person is discarded, and is placed in sterile chamber, is put less than -20 DEG C freezen protectives.It is labelled per bottle embryo liquid, and
Indicate the harvest date.
2nd, antigen detection
2.1 steriling test
By existing《Chinese veterinary pharmacopoeia》Annex is tested.
2.2 viral levels determine
Seed culture of viruses physiological saline work is serially diluted for 10 times, takes 10-4、10-5、10-6、10-74 dilution factors, each dilution
Degree is inoculated with 12 5 pieces of age in days goose embryos through allantoic cavity, per embryo 0.2ml, puts dead goose embryo before 37 DEG C of incubations are observed 168 hours, 72 hours
Disregard, record 72~168 hours goose embryo death conditions, calculate ELD50, >=10 are answered per 0.2ml viral levels5.0ELD50。
3rd, the concentration and inactivation of antigen
Qualified goose blastochyle will be examined to be mixed in same container.Ultrafiltration by supernatant through molecular cut off for 50kDa
Film bag carries out 10 times of concentrations, and the antigen after concentration adds final concentration of 0.2% formalin (percent by volume), shakes up, and seals
37 DEG C are put to inactivate 48 hours (6~8 hours periods shook 1 time).
4th, inactivation antigen is examined
The antigen after inactivation is taken, susceptible 5 pieces of the goose embryo of 12 ages in days is inoculated with through allantoic cavity, per embryo 0.2ml, puts 37 DEG C of cultures 168
Hour, goose embryo should all be good for work.
5th, the preparation of immunogene
It is prepared by 5.1 aqueous phases
Count by volume, qualified concentrated antigen 96 parts of additions, 4 parts of Tween-80s will be examined, make Tween-80 after shake well
Untill being completely dissolved.
It is prepared by 5.2 oil phases
Count by volume, take 94 parts of injection white oil, add 1.5 parts of aluminum stearate, it is stirring while adding, until completely thoroughly
It is bright, 6 parts of Si Ben -80 is added, it is standby fully to mix autoclaving.
5.3 emulsification
Aqueous phase and oil phase 1:1.5 ratio (V:V), oil phase is injected in premixing tank, 2500 revs/min of low temperature stirrings, delayed
It is slow to add aqueous phase, emulsion tank emulsification is reinjected with 10000 revs/min, is emulsified 5 minutes.
5.4 packing
Sterile quantitative separating, bottleneck is sealed, puts 2~8 DEG C of preservations.
6th, immunogene is examined
6.1 physical behavior
Outward appearance:Milky emulsion.
Formulation:Water-in-oil type.A cleaning suction pipe is taken, a small amount of vaccine is drawn and instills in cold water, all should oil droplet in addition to first drips
Shape indiffusion.
Stability:Draw immunogene 10ml to add in centrifuge tube, centrifuged 15 minutes with 3000r/min, the water that ttom of pipe separates out
Accordingly≤0.5ml.
Viscosity:By existing《Chinese veterinary pharmacopoeia》Annex is tested, and should meet regulation.
6.2 steriling test
By existing《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
6.3 safety verification
Take 28 age in days SPF chickens 10, every subcutaneously or intramuscularly multi-point injection immunogene 2.0ml, observe 14, should all be good for
It is living.
7th, it is immunized
Head is immunized based on exempting from, chest muscle multi-point injection gosling plague immunogene 1.0ml and Radix Astragali polysaccharide injection
1.0ml, it is spaced progress two on the 14th and exempts from booster immunization, chest muscle multi-point injection gosling plague immunogene 2.0ml and astragalus polyose note
Liquid 1.0ml is penetrated, progress three on the 14th is spaced and exempts from reinforced immunological, chest muscle multi-point injection gosling plague immunogene 3.0ml and the Radix Astragali are more
Sugared parenteral solution 1.0ml.
8th, positive serum separation and preparation
Sterile Culling heart blood on the 14th after reinforcement is exempted from, put 37 DEG C and act on 1 hour, 4 DEG C act on 1 hour, sterile separation serum.And
Dispensed by 1.0ml/ bottles, chilled vacuum drying is made, and mark Serum information, lot number, puts -20 DEG C of preservations.
9th, gosling plague positive serum is examined
9.1 characters are examined
This product should be the faint yellow or loose agglomerate of pale red, be dissolved rapidly after adding dilution.
9.2 steriling test
By existing《Chinese veterinary pharmacopoeia》Annex is tested, and should meet regulation.
9.3 specific assay
Antibody is randomly selected, is exhaled with gosling plague, infections chicken cloacal bursa, ewcastle disease, Marek's disease, fowl A types influenza, fowl
The lonely disease of intestines, chicken pox agp antigen carry out agar gel diffusion test, and result was observed in 24~48 hours, precipitation line should not occur.
9.4 titration
Antibody is randomly selected, 2 times is done with physiological saline and is serially diluted, takes 8 times, 16 times, 32 times, 64 times of 4 dilution factors, with
Gosling plague agp antigen carries out agar gel diffusion test.Result was observed in 24~48 hours, potency should be not less than 1:32.
9.5 residual moisture
By existing《Republic of China Veterinary Pharmacopoeia》Annex is measured, and should meet regulation.
Gosling plague positive serum prepares and examines technological process to see Fig. 2.
The gosling plague negative serum of embodiment 4 is prepared and examined
1st, negative serum separation and preparation
Selection gosling plague antibody is negative into goose, sterile Culling heart blood, puts 37 DEG C and acts on 1 hour, 4 DEG C act on 1 hour, nothing
Bacterium separates serum.And dispensed by 1.0ml/ bottles, chilled vacuum drying is made, and mark Serum information, lot number, puts -20 DEG C of preservations.
2nd, product inspection
2.1 characters are examined
This product should be the faint yellow or loose agglomerate of pale red, be dissolved rapidly after adding dilution.
2.2 steriling test
By existing《Chinese veterinary pharmacopoeia》Annex is tested, and should meet regulation.
2.3 specific assay
Antibody is randomly selected, is exhaled with gosling plague, infections chicken cloacal bursa, ewcastle disease, Marek's disease, fowl A types influenza, fowl
The lonely disease of intestines, chicken pox agp antigen carry out agar gel diffusion test, observe result in 24~48 hours, all should occur without any precipitation
Line.
2.4 titration
Antibody is randomly selected, after being diluted by labelled amount, agar gel diffusion test is carried out with gosling plague agp antigen.24~48
Observation result in hour, it should be negative.
2.5 residual moisture
By existing《Republic of China Veterinary Pharmacopoeia》Annex is measured, and should meet regulation.
Gosling plague negative serum prepares and examines technological process to see Fig. 3.
Claims (11)
- A kind of 1. gosling plague antibody test standard substance, it is characterised in that including:Gosling plague agp antigen, gosling plague positive blood Cleer and peaceful gosling plague negative serum.
- 2. according to the gosling plague antibody test standard substance described in claim 1, it is characterised in that the gosling plague agp antigen Preparation include:(1) goose parvovirus strain is inoculated with susceptible goose embryo, harvests goose blastochyle, obtain antigen liquid；(2) to antigen liquid Middle addition freeze drying protectant, mix, freeze-drying, produce.
- 3. according to the gosling plague antibody test standard substance described in claim 2, it is characterised in that step (1) described antigen liquid Viral level be:Per viral level >=10 of 0.2ml antigen liquids5.0ELD50；Step (1) the goose parvovirus strain is preferably goose parvovirus H strains, and its microbial preservation numbering is:CGMCC No.6851；Step (1) the goose blastochyle is to be incubated the goose blastochyle of goose embryo dead in 72-168 hours.
- 4. according to the gosling plague antibody test standard substance described in claim 2, it is characterised in that step (2) is according to volume ratio Meter, antigen liquid:Freeze drying protectant=1-3:1-3, preferably 1:1；Preferably, the freeze drying protectant is made up of sucrose, skimmed milk and water；It is furthermore preferred that based on g/ml, the frozen-dried protective Final concentration of the 2.5% of sucrose in agent, final concentration of the 2.5% of skimmed milk, surplus is water.
- 5. according to the gosling plague antibody test standard substance described in claim 1, it is characterised in that the gosling plague positive serum Preparation include:(1) goose parvovirus strain is inoculated with susceptible goose embryo, harvests goose blastochyle, obtain antigen liquid；(2) by antigen liquid Concentration, inactivation, obtained inactivation antigen prepare gosling plague immunogene；(3) by the susceptible goose of gosling plague immunogen immune health, adopt Blood, serum is separated, freeze-drying, is produced；Antibody titer >=1 of the gosling plague positive serum:32.
- 6. according to the gosling plague antibody test standard substance described in claim 5, it is characterised in that step (1) described goose is tiny Virus stain is preferably goose parvovirus H strains, and its microbial preservation numbering is:CGMCC No.6851；The goose blastochyle is incubation The goose blastochyle of dead goose embryo in 72-168 hours；The viral level of the antigen liquid is:Per the viral level of 0.2ml antigen liquids ≥105.0ELD50；Step (2) concentration is that antigen liquid is carried out into 10 times of concentrations；Preferably, the concentration includes:Antigen liquid is centrifuged, taken Supernatant, the milipore filter bag for being 50kDa with molecular cut off carry out 10 times of concentrations；The inactivation includes:Count by volume, to dense Final concentration of 0.2% formalin is added in antigen after contracting, is mixed, 37 DEG C inactivate 48 hours.
- 7. according to the gosling plague antibody test standard substance described in claim 5, it is characterised in that step (2) described gosling plague The preparation of immunogene includes:(a) inactivation antigen is well mixed with Tween-80, obtains aqueous phase；(b) by injection white oil, tristearin Sour aluminium is well mixed with Si Ben -80, sterilizing, obtains oil phase；(c) aqueous phase is mixed with oil phase, emulsifies, produce.
- 8. according to the gosling plague antibody test standard substance described in claim 7, it is characterised in that step (a) is counted by volume, Inactivation antigen:Tween-80=96:4；Step (b) is counted by volume, injection white oil:Aluminum stearate:Si Ben -80=94:1.5:6；Step (c) is counted by volume, aqueous phase:Oil phase=1:1.5.
- 9. according to the gosling plague antibody test standard substance described in claim 5, it is characterised in that step (3) is described immune Program includes:Fundamental immunity, carry out booster immunization within 14 days after fundamental immunity, carry out reinforced immunological within 14 days after booster immunization；Wherein, the dosage of inoculation of the fundamental immunity is:Gosling plague immunogene 1.0-2.0ml and Radix Astragali polysaccharide injection 1.0ml； The dosage of inoculation of the booster immunization is:Gosling plague immunogene 1.0-3.0ml and Radix Astragali polysaccharide injection 1.0ml；The reinforcing Immune dosage of inoculation is:Gosling plague immunogene 1.0-3.0ml and Radix Astragali polysaccharide injection 1.0ml；Preferably, the dosage of inoculation of the fundamental immunity is:Gosling plague immunogene 1.0ml and Radix Astragali polysaccharide injection 1.0ml；Institute The dosage of inoculation for stating booster immunization is:Gosling plague immunogene 2.0ml and Radix Astragali polysaccharide injection 1.0ml；The reinforced immunological Dosage of inoculation is:Gosling plague immunogene 3.0ml and Radix Astragali polysaccharide injection 1.0ml；The immune route of inoculation is intramuscular injection, preferably chest muscle multi-point injection；The content of astragalus polyose is 20mg/ml in the Radix Astragali polysaccharide injection；Step (3) is described to take a blood sample to be taken a blood sample within 14th after reinforced immunological.
- 10. according to the gosling plague antibody test standard substance described in claim 1, it is characterised in that the gosling plague feminine gender blood Clear preparation includes:What gosling plague antibody was negative takes a blood sample into goose, separates serum, freeze-drying, produces.
- 11. the gosling plague antibody test standard substance described in claim 1 to 10 any one is preparing the examination of detection gosling plague Application in agent.
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