CN107304230A - A kind of anti-dog parvovirus refines antibody and preparation method thereof - Google Patents
A kind of anti-dog parvovirus refines antibody and preparation method thereof Download PDFInfo
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- CN107304230A CN107304230A CN201710596570.5A CN201710596570A CN107304230A CN 107304230 A CN107304230 A CN 107304230A CN 201710596570 A CN201710596570 A CN 201710596570A CN 107304230 A CN107304230 A CN 107304230A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Abstract
Antibody and preparation method thereof is refined the invention discloses a kind of anti-dog parvovirus.Comprise the following steps：S1. strain prepares canine parvovirus based on canine parvovirus the CPV S5 and CPV 1401 for separating identification by In Guangdong Province first；S2. vaccine immunity Healthy Dogs only prepare canine parvovirus hyper-immune serum；S3. the anti-dog parvovirus immunoglobulin in the serum that prepared by saturated ammonium sulfate substep salting-out separation S2；S4. purify：The anti-dog parvovirus immunoglobulin that sephadex is extracted to step S3 carries out desalination；S5. by the anti-dog parvovirus immunoglobulin ultrafiltration concentration after step S4 desalting processings, filtration sterilization, packing, produce.Anti- anti-dog parvovirus prepared by the present invention refines antibody protein content and reaches 134mg/ml, and blood clotting suppresses potency and is not less than 1：10240；The treatment and urgent prevention when each kind dog is infected by contact with parvovirus, the ill dog immunity of organisms of increase and resistance are can be applied to, is had no adverse reaction, no clinical side effects, security is good, with good popularizing application prospect.
Description
Technical field
The invention belongs to biopharmaceutical technology.More particularly, to a kind of anti-dog parvovirus refine antibody and its
Preparation method.
Background technology
Canine parvovirus disease, also known as canine parvoviral enteritis, are by canine parvovirus（Canine parbovirus,
CPV）A kind of caused highly contagious disease, mainly causes dog acute hemorrhagic entertitis or Neonatal Canine acute myocarditis, hair
The clinical symptoms of sick dog are mainly that body temperature rise, hyperemesis, hemorrhagic enteritis and leucocyte are substantially reduced.The disease is throughout the year equal
Hair, especially in the majority with spring and autumn, variety classes, the dog at age can infect this disease, wherein endanger maximum with pup, infection
Rate and death rate highest, seriously endanger China's canine farming.At present, it is certain to prevent the disease to obtain to use vaccine immunity injecting method
Effect, but because of reasons such as maternal antibody, strain variations, often immuning failure occur and infect canine parvovirus.CPV only one of which
Antigen serotype CPV-2, but due to antigenic drift, the change of VP2 genes causes amino acid substitution to occur in that different genes are sub-
Type, the CPV-2c that CPV-2a, CPV-2b and 2000 are found from Italy mutant, these new subtypes have possibility
It is one of the reason for causing immuning failure.Huang Yongliang（2012）In Shenzhen（9 plants）And Guangzhou（10 plants）19 plants of CPV are assigned in area,
Identify that only 1 plant is CPV-2b, 2 plants of New CPV-2b through separation, remaining is New CPV-2a hypotypes.This shows in Guangzhou and depth
Ditch between fields popular strain by advantage of New CPV-2a.In terms for the treatment of, effectively preventing canine parvovirus cytotoxic drug and prevention and controls are there is no,
So, the cure rate of canine parvovirus is low.Anti-dog parvovirus immunoglobulin concentrate formulation is preventing and treating canine parvovirus disease
Specific biological product, is a kind of passive immunity preparation, while the effect of also urgent immunization campaign, should with higher popularization
With value.
Immunoglobulin (Immunoglobulin, abbreviation Ig) be body by after antigenic stimulus, be particularly by lymphocyte
One class of plasma cell synthesis has the globulin of antibody activity.Immunoglobulin is prevalent in the blood of mammal, tissue
In liquid, lymph and exocrine secretion.Immunoglobulin has important immune and physiological regulatory action in animal body, is animal
One of the most key component of vivo immuning system.Its topmost biological characteristics is exactly and corresponding antigentic specificity
With reference to also activating complement promotees the functions such as phagocytosis.The use of immunoglobulin, it is passive to can be described as in Immunology
It is immune, when immune deficiency, vaccine shortage and immuning failure, Active immunisation, hair are substituted using immunoglobulin injection
Wave resistant effect.
Therefore, one species specificity of exploitation is for the highly concentrated of canine parvovirus New CPV-2a and New two kinds of hypotypes of CPV-2b
The anti-dog parvovirus immunoglobulin preparation of degree, high activity and high-titer, is clinical treatment and urgent prevention canine parvovirus
The effective means of disease.
The content of the invention
The technical problem to be solved in the present invention be overcome existing canine parvovirus disease protective agents defect and it is not enough there is provided
A kind of medicine --- anti-dog parvovirus immunoglobulin preparation for preventing and treating canine parvovirus disease, i.e., a kind of anti-dog parvovirus essence
Antibody processed.
Antibody is refined it is an object of the invention to provide a kind of anti-dog parvovirus.
Another object of the present invention is to provide the preparation method that the anti-dog parvovirus refines antibody.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of anti-dog parvovirus refines the preparation method of antibody, comprises the following steps：
S1. canine parvovirus is prepared；
S2. vaccine immunity Healthy Dogs only prepare canine parvovirus hyper-immune serum；
S3. the anti-dog parvovirus immunoglobulin in the serum that prepared by saturated ammonium sulfate substep salting-out separation S2；
S4. purify：The anti-dog parvovirus immunoglobulin that sephadex is extracted to step S3 carries out desalination；
S5. by the anti-dog parvovirus immunoglobulin ultrafiltration concentration after step S4 desalting processings, filtration sterilization, packing, produce.
Wherein it is preferred to, step S1 method is：Canine parvovirus CPV-S5 (the New of identification are separated with In Guangdong Province
CPV-2a strain) and based on CPV-1401 (New CPV-2b), expands culture, small-sized slipstream on F81 cells and surpasses respectively
After filter concentration 9 are pressed with water-soluble adjuvant MONTANIDE GEL：1 ratio is mixed with inactivated vaccine.
Preferably, step S2 method is：The negative Healthy Dogs of 5 week old CPV HI are chosen only, while dorsal sc multiple spot
It is inoculated with canine parvovirus two kinds of inactivated vaccines of CPV-S5 and CPV-1401,25~30d（It is preferred that 28d）Add afterwards according to same method
It is strong immune；7~14d after immune, arteria carotis aseptic collection CPV HI antibody titers are not less than 1：3072 dog blood, stands, centrifuges
Isolated serum.
Preferably, the immunizing dose of two kinds of inactivated vaccines of canine parvovirus CPV-S5 and CPV-1401 is 1mL/.
Preferably, the step of step S3 saturated ammonium sulfates substep is saltoutd is as follows：
（a）By serum phosphate buffer（PBS）Precooling saturated ammonium sulfate is slowly added dropwise under dilution, magnetic agitation（AS）Solution is extremely
Final concentration of 15~25%, 10~30min is stirred, 0~10 DEG C stands after 0.5~2h, 5000~8000r/min centrifugations 20~
40min, abandons precipitation；
（b）To step（a）In obtained supernatant solution, precooling saturated ammonium sulfate solution is slowly added dropwise under magnetic agitation to final concentration
40~60%, 0~4 DEG C stands after 0.5~2h, and 5000~8000r/min centrifuges 2~40min, abandons supernatant, collects precipitation；
（c）To step（b）The PBS that former serum two volumes are added in gained precipitation dissolves, and is slowly added dropwise under magnetic stirring apparatus pre-
Cold saturation AS solution is to final concentration 25%~35%, and 0~4 DEG C stands 0.5~2h, and 5000~8000r/min centrifuges 20~40 min,
Supernatant is abandoned, precipitation is collected；
（d）Repeat step（c）Twice.
The step of step S3 saturated ammonium sulfates substep is saltoutd is most preferably as follows：
（a）By serum phosphate buffer（PBS, pH=7.0）Make that precooling saturation sulphur is slowly added dropwise under 2 times of dilutions, magnetic agitation
Sour ammonium（AS）Solution（pH = 7.0）To final concentration of 20%, 20min is stirred, 4 DEG C stand after 1 h, 6500 r/min centrifugations
30min, abandons precipitation；
（b）To step（a）In obtained supernatant solution, precooling saturated ammonium sulfate is slowly added dropwise under magnetic agitation（AS）Solution（pH=
7.0）To final concentration 50%, 4 DEG C stand after 1 h, 6500 r/min centrifugation 30min, abandon supernatant, collect precipitation；
（c）To step（b）The PBS that former serum two volumes are added in gained precipitation dissolves, and is slowly added dropwise under magnetic stirring apparatus full
With AS solution to final concentration 33%, 4 DEG C of standings 1 h, 6500 r/min centrifuge 30 min, abandon supernatant, collect precipitation；
（d）Repeat step（c）Twice.
Preferably, the method purified described in step S4 is, using Sephadex G-25 prepackage gel columns, to be gathered using Portugal
The anti-dog parvovirus immunoglobulin that sugared gel is extracted to step S3 carries out desalting and purifying.Comprise the following steps that：
（1）Prepacked column is connected with protein purification system, during prevent air from entering cylinder；
（2）Balance pillar：Pillar is balanced with the PBS of 5 times of column volumes, with the complete ethanol for removing preservation pillar, while so that
Pillar is filled up completely with PBS, and flow control is in 5 mL/min；
（3）Loading and elution：Continue to be filled with PBS elutions after sample volume maximum 1.5 mL, flow velocity 1mL/min, completion of the sample；
（4）Collect：Using ultraviolet absorption value, resistivity as reference, eluent at peak is collected with 1.5 mL ep pipes, -20 DEG C of preservations are standby
With；
（5）Wash pillar：Washed, then washed with 20% ethanol of 5 times of column volumes with the PBS of 5 times of column volumes, to preserve pillar.
Preferably, it is concentrated by ultrafiltration to concentrate using small-sized cross-flow ultrafiltration described in step S5, it is 1 to produce HI potency：10240
Refined antibody.
In order to obtain that protein content is higher, antibody activity more preferably and the lower immunoglobulin of endotoxin content,
The method that the present invention separated and extracted the immunoglobulin in serum to traditional saturated ammonium sulfate is optimized.As a result table
It is bright, use the anti-dog parvovirus prepared by the saturated ammonium sulfate after optimization to refine the immunoglobulin concentrations of antibody for 134
mg/mL；The measurement result of CPV HI potency shows, using the anti-canine parvovirus prepared by the saturated ammonium sulfate salting out method after optimization
Poison refines antibody titer up to 1:10240.
Most preferably, the preparation method of the refined antibody of canine parvovirus (CPV) of the present invention is：Will be with In Guangdong Province
Based on separating the canine parvovirus CPV-S5 (New CPV-2a) and CPV-1401 (New CPV-2b) of identification prepared by strain
Two kinds of inactivated vaccines, the negative dogs of CPV are only subcutaneously injected according to 1mL/ only, booster immunization after 28 d.Two exempt from rear 7 d HI antibody
Reach peak.Two exempt from 10 d, and arteria carotis is sterile to take blood, and HI potency is obtained after mixing and is not less than 1：3072 hyper-immune serum.Adopt
Saltoutd with 20%~50% saturated ammonium sulfate substep and the thick most of foreign protein of purification removing is carried out to hyper-immune serum, to salt precipitation
Ammonium sulfate concentrations, pH are optimized, and it is optimal conditions to determine saturated ammonium sulfate concentration 33%, pH=7.0.It is according to said method a large amount of to extract
After purification, through HiTrap Desalting 5mL prepacked column desalinations, then it is obtained anti-by small-sized cross-flow ultrafiltration concentration systems
Canine parvovirus refines antibody, and HI potency is up to 1：10240.
To sum up, using prepared by the saturated ammonium sulfate salting out method after optimization anti-dog parvovirus refine antibody concentration,
Purity is of a relatively high；Canine parvovirus blood clotting suppresses potency and significantly improved.
In addition, the anti-dog parvovirus prepared according to the above method refines antibody and its application also in the guarantor of the present invention
Within the scope of shield.
The anti-dog parvovirus, which refines antibody, includes anti-dog parvovirus immunoglobulin；Wherein, the anti-dog is tiny
Content ＞ 130 mg/mL, CPV the HI antibody titers of virus immunity globulin are up to 1：More than 10240.
Anti-dog parvovirus prepared by the present invention refines the liquid or freeze-dried formulation that antibody is pH7.0 or so；Wherein, anti-dog
It is main with the presence of IgG monomers that parvovirus refines antibody.Anti-dog parvovirus of the present invention refines antibody long shelf-life, and 2~8 DEG C can
Preserve more than 2 years.
Anti-dog parvovirus of the present invention, which is refined in antibody, can also add suitable protective agent, such as maltose, glucose
Deng.Those skilled in the art can be adjusted to protectant consumption according to actual needs.
In addition, anti-dog parvovirus antibody of the present invention can apply to prevent and treat canine parvovirus disease, moreover it is possible to
It is enough to be applied to prevent or treatment canine parvovirus viral enteritis, ill dog immunity of organisms and resistance can be increased.
Therefore, the anti-dog parvovirus for preparing of the present invention refine antibody as or prepare the prevention of canine parvovirus disease
And/or the application in terms of medicine, as or prepare prevent and/or treatment canine parvovirus viral enteritis medicine
Or the application in terms of reagent, as or prepare can increase ill dog（The ill dog refers to the dog for suffering from canine parvovirus disease）
Application in immunity of organisms and the medicine of resistance, all should be within protection scope of the present invention.
Anti-dog parvovirus of the present invention refines the application method of antibody：General use subcutaneously or intramuscularly is injected；Mainly
For the treatment and urgent prevention of the parvovirus infections of each kind dog, ill dog immunity of organisms and premunition are effectively increased.
The invention has the advantages that：
Anti- anti-dog parvovirus prepared by the present invention refines antibody protein content and reaches 134mg/ml, and blood clotting suppresses potency and is not less than
1：10240；It can be applied to the treatment and urgent prevention when each kind dog is infected by contact with parvovirus, the ill dog immunity of organism of increase
Power and resistance.
Moreover, safety experiment assay shows, antibody preparation inoculation dog is only（Such as to dog muscle or subcutaneous note
Penetrate）Afterwards, dog spirit, body temperature, appetite are normal, and injection site does not have lump, has no adverse reaction, no clinical side effects, safety
Property is good, with good popularizing application prospect.
Anti- anti-dog parvovirus after present invention optimization refines the preparation method of antibody, both maintains immunoglobulin molecules
The integrality and bioactivity of structure, while agents useful for same is cheap, simple to operate in preparation process, improve lipidated protein,
Immunoglobulin content and biological safety.
Brief description of the drawings
Fig. 1 is that different AS concentration extract protein SDS-PAGE analysis；M. standard protein.
Fig. 2 is that anti-dog parvovirus refines antibody and former serum SDS-PAGE analyses；1. the anti-dog of hyper-immune serum 2. is tiny
The refined antibody of virus, M. albumen marker.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
The term definition that the present invention relates to：
Unless otherwise defined, otherwise all technologies used herein and scientific terminology all have with it is of the art common
Technical staff generally understands identical implication.
Term " test by erythrocyte agglutination（HA）Suppress to test with erythrocyte agglutination（HI）", canine parvovirus has spy at 4 DEG C
The effect of different in nature aggegation swine erythrocyte, referred to as erythrocyte agglutination are tested.Blood clotting phenomenon can be suppressed to be referred to as red blood cell by corresponding antibodies
Agglutination inhibition test, so that the highest serum dilution factor of 100% erythrocyte agglutination can be suppressed as the HI potency of the serum.
The anti-dog parvovirus of embodiment 1 refines the preparation of antibody
1. test method
The preparation of 1.1 anti-dog parvovirus hyper-immune serums
CPV-S5 (New CPV-2a) and CPV-1401 (New CPV-2b) two are prepared according to conventional inactivated vaccine preparation method
Strain inactivated vaccine.Choose the negative healthy pup of a number of CPV HI potency, dorsal sc multiple spot inoculation CPV-S5 and
Two kinds of inactivated vaccines of CPV-1401, treat that HI potency drops to 1:Less than 80（4 weeks or so）Carry out booster immunization.
Two exempt from 10 ~ 14 d after the completion of inoculation, with hemagglutination-inhibition test（HI）Antibody titer is determined, antibody titer is chosen not
Less than 1：2048 dog, arteria carotis sterile blood sampling, after mixing, Cord blood.
1.2 saturated ammonium sulfate（AS）Substep is saltoutd
（1）The serum of above-mentioned preparation is melted, the phosphate buffer of equimultiple volume is added（PBS, pH=7.0）, under magnetic agitation
Saturated ammonium sulfate is slowly added dropwise（AS, pH=7.0）Solution stirs 20 min to final concentration of 20%, and 4 DEG C of refrigerators stand 1 h, taken
6500 r/min centrifuge 30 min after going out, and harvest supernatant.
（2）To step under magnetic agitation（1）Saturation AS solution is slowly added dropwise in the supernatant of harvest to final concentration 50%, stirs
20 min, 4 DEG C of 1 h of standing are mixed, 6500 r/min centrifuge 30 min after taking-up, abandon supernatant, collect precipitation.
（3）With the PBS dissolving steps of former serum two volumes（2）Saturation is slowly added dropwise under obtained precipitation, magnetic agitation
AS solution is to different final concentrations（25%~35%）, 20 min are stirred, 4 DEG C of refrigerators stand 1 h, and 6500 r/min are centrifuged after taking-up
30 min, abandon supernatant, collect precipitation.
（4）Repeat step（3）Twice.
The mL prepacked column desalinations of 1.3 HiTrap Desalting 5
（1）Prepacked column is connected with protein purification system, during prevent air from entering cylinder.
（2）Balance pillar：Pillar is balanced with the PBS of 5 times of column volumes, the ethanol of pillar is preserved with complete removing, simultaneously
So that pillar is filled up completely with PBS, flow control is in 5 mL/min.
（3）Loading and elution：Continue to be filled with PBS after sample volume maximum 1.5 mL, the mL/min of flow velocity 1, completion of the sample and wash
It is de-.
（4）Collect：Using ultraviolet absorption value, resistivity as reference, eluent at peak, -20 DEG C of guarantors are collected with 1.5 mL ep pipes
Deposit standby.
（5）Wash pillar：Washed, then washed with 20% ethanol of 5 times of column volumes with the PBS of 5 times of column volumes, to preserve post
Son.
1.4 are concentrated by ultrafiltration immunoglobulin
（1）By milipore filter（Pellicon XL Cassette, Biomax 50kDa）By specification and small-sized cross-flow ultrafiltration machine
Device is connected.
（2）500 mL tri-distilled waters are added into sample cell, small-sized cross-flow ultrafiltration system is cleaned, circulation 20
min。
（3）Distilled water is discharged, 500 mL 0.1M NaOH solution is added and small-sized cross-flow ultrafiltration system is disappeared
Poison, circulates 20 min.
（4）Discharge NaOH solution, repeat step（2）.
（5）Distilled water is discharged, the 200 mL min of PBS rinses 10 is added, discharges afterwards.
（6）Loading：Add sample to be concentrated（A diameter of 0.45 μm of membrane filtration）, pressure valve is adjusted, pressure is prevented
Power is excessive to cause film to damage.Whole process keeps low temperature, prevents virally inactivated.
（7）Concentration volume is reached, sample is exported, sterile centrifugation tube packing, -80 DEG C save backup.
（8）Repeat step（3）→（2）→（3）, film be full of fresh 0.1M NaOH solution after, pull down, 4 DEG C preservation.
1.5 anti-dog parvovirus refine antibody protein concentration and purity detecting
Purity of protein is analyzed using BCA methods detection protein concentration, through SDS-PAGE protein electrophoresises.
2. experimental result
Fig. 1 is that different AS concentration extract protein SDS-PAGE analysis result.Fig. 2 is that anti-dog parvovirus refines antibody and former serum
SDS-PAGE comparative analysis results.
The protein concentration that anti-dog parvovirus prepared by the present invention refines antibody is 134 mg/mL, and blood clotting suppresses potency
（HI）For：12800.
The refined antibody safety detection of the anti-dog parvovirus of embodiment 2 virus
1. experimental method
Press respectively per the mL dosage of kg body weight 1.0,2.0,4.0, subcutaneous, 5 monthly age of intramuscular injection beasle dog, the h of Continuous Observation 48,
Continue breeding observing 3 months, record experiment dog injection site whether there is swelling, whether allergy, spirit and diet.
2. experimental result
By the beasle dog of the refined antibody of the anti-anti-dog parvovirus of various dose injection, the h of Continuous Observation 48, injection site has no
The anomaly such as swelling and allergy, body temperature and spiritual diet are normal.Continue to raise 3 months, during which also without any different
Often, show that anti-anti-dog parvovirus refines antibody and had no side effect, be adapted to clinical expansion.
Claims (10)
1. a kind of anti-dog parvovirus refines the preparation method of antibody, it is characterised in that comprise the following steps：
S1. canine parvovirus is prepared；
S2. vaccine immunity Healthy Dogs only prepare canine parvovirus hyper-immune serum；
S3. the anti-dog parvovirus immunoglobulin in the serum that prepared by saturated ammonium sulfate solution substep salting-out separation S2；
S4. purify：The anti-dog parvovirus immunoglobulin that sephadex is extracted to step S3 carries out desalination；
S5. by the anti-dog parvovirus immunoglobulin ultrafiltration concentration after step S4 desalting processings, filtration sterilization, packing, produce.
2. preparation method according to claim 1, it is characterised in that step S1 method is：Separated and reflected with In Guangdong Province
Strain based on fixed canine parvovirus CPV-S5 and CPV-1401, expands culture, small-sized slipstream on F81 cells and surpasses respectively
After filter concentration 9 are pressed with water-soluble adjuvant MONTANIDE GEL：1 ratio is mixed with inactivated vaccine.
3. preparation method according to claim 1, it is characterised in that step S2 method is：Choose 5 week old CPV HI
Negative Healthy Dogs, while dorsal sc multiple spot is inoculated with canine parvovirus two kinds of inactivated vaccines of CPV-S5 and CPV-1401,25
According to same method booster immunization after~30d；7~14d after immune, arteria carotis aseptic collection CPV HI antibody titers are not less than 1：
3072 dog blood, stands, is centrifugally separating to obtain serum.
4. preparation method according to claim 1, it is characterised in that the step that step S3 saturated ammonium sulfates solution substep is saltoutd
It is rapid as follows：
（a）Serum is diluted with phosphate buffer, precooling saturated ammonium sulfate solution is slowly added dropwise under magnetic agitation to final concentration of
15~25%, 10~30min is stirred, 0~10 DEG C stands after 0.5~2h, and 5000~8000r/min centrifuges 20~40min, abandons heavy
Form sediment；
（b）To step（a）In obtained supernatant solution, precooling saturated ammonium sulfate solution is slowly added dropwise under magnetic agitation to final concentration
40~60%, 0~4 DEG C stands after 0.5~2h, and 5000~8000r/min centrifuges 2~40min, abandons supernatant, collects precipitation；
（c）To step（b）The PBS that former serum two volumes are added in gained precipitation dissolves, and is slowly added dropwise under magnetic stirring apparatus pre-
Cold saturation AS solution is to final concentration 25%~35%, and 0~4 DEG C stands 0.5~2h, and 5000~8000r/min centrifuges 20~40 min,
Supernatant is abandoned, precipitation is collected；
（d）Repeat step（c）Twice.
5. preparation method according to claim 1, it is characterised in that the method purified described in step S4 is use
Sephadex G-25 pre-install gel column, the anti-dog parvovirus immune globulin extracted using sephadex to step S3
It is white to carry out desalting and purifying.
6. preparation method according to claim 1, it is characterised in that it is using small-sized tangential to be concentrated by ultrafiltration described in step S5
Stream is concentrated by ultrafiltration.
7. the anti-dog parvovirus prepared according to any methods described of claim 1~6 refines antibody.
8. anti-dog parvovirus refines antibody according to claim 7, it is characterised in that immune including anti-dog parvovirus
Globulin；Wherein, content ＞ 130 mg/mL, CPV the HI antibody titers of the anti-dog parvovirus immunoglobulin are not less than
1：10240.
9. anti-dog parvovirus described in claim 7 refine antibody as or prepare the prevention of canine parvovirus disease and/or control
Treat medicine in terms of application, as or prepare prevent and/or treatment canine parvovirus viral enteritis medicine or reagent
The application of aspect.
10. anti-dog parvovirus described in claim 7 refine antibody as or prepare can increase ill dog immunity of organisms and
Application in the medicine of resistance, the ill dog refers to the dog for having infected canine parvovirus.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110862451A (en) * | 2019-11-29 | 2020-03-06 | 天津瑞普生物技术股份有限公司 | Preparation method and application of equine anti-canine parvovirus immunoglobulin |
| CN112079916A (en) * | 2020-02-28 | 2020-12-15 | 衡阳师范学院 | Canine parvovirus egg yolk antibody and preparation method thereof |
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