CN105884889A - Canine distemper/infectious hepatitis/parvovirus triple egg yolk antibody and preparation method thereof - Google Patents
Canine distemper/infectious hepatitis/parvovirus triple egg yolk antibody and preparation method thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
-
- 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
-
- 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
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
Abstract
The invention provides a preparation method of a canine distemper/infectious hepatitis/parvovirus triple egg yolk antibody and provides the canine distemper/infectious hepatitis/parvovirus triple egg yolk antibody prepared with the preparation method. The preparation method comprises steps as follows: eggs laid by immunized layer hens are collected and subjected to surface sterilization, egg yolk is separated, homogenized and diluted with sterile distilled water, pH is regulated to 5.0-5.2, a mixture is left to stand, and a supernatant is collected; the supernatant is diluted with an acetate buffer solution, pH is regulated to 5.0-5.2, n-caprylic acid is added, a mixture is left to stand for layering, a water phase is taken and centrifuged, precipitates are taken and dissolved in normal saline, filtering is performed, and the egg yolk antibody is obtained. The egg yolk antibody has the advantages of stable physicochemical properties, wide sources, high output and low cost as well as large animal phylogenetic distance, is more suitable for producing specific antibodies, can be used for preparing a diagnostic reagent, controlling diseases and serving as a feed additive, and has the potential in development of functional foods and new drugs.
Description
Technical field
The present invention relates to biological field, specifically, relate to a kind of canine distemper-infectious hepatitis-parvovirus three
Yolk antibody and preparation method thereof.
Background technology
Canine distemper virus, canine infectious hepatitis virus and canine parvovirus disease are all the most common dog viroid property
Infectious disease, although every country has increased the immune prevention and control to these three disease now, but due to these three disease
High degree in contact, the feature such as lethal so that these three disease is the most popular, and cause huge economy
Loss.Canine distemper is to be caused by canine distemper virus (Canine distemper virus, CDV), and its main clinic symptoms is:
Body temperature in " diphasic fever " change, pneumonia, rhinorrhea, have gum etc.；Infectious canine hepatitis is by dog 1 type adenovirus (Canine
Adenovirus virus type l, CAV-l) have another name called canine infectious hepatitis virus (Canine infectious
Hepatitis virus, ICHV) cause, main clinic symptoms for vomitting, suffer from abdominal pain, suffering from diarrhoea, body temperature rising, sudden death etc.；
Canine parvovirus disease is to be caused, its main clinic symptoms by Canine Parvovirus (Canine parvovirus virus, CPV)
For vomiting, draw Fructus Lycopersici esculenti sample of blood dysentery etc..
Antibody (immunoglobulin, Immunoglobulin, Ig) refer to body by after exotic antigen material incentive, B is thin
Born of the same parents identify that antigen is postactivated, proliferation and differentiation is dress cell, by dress cell synthesis and secretion can be with corresponding antigens specific recognition
With the class globulin combined.Hen after specific antigen immunity can produce corresponding specific antibody, has research to confirm, produces
The yolk antagonist of laying hen has concentration effect, makes the antibody in serum constantly be stored in the yolk of growth, and this antibody is referred to as
Yolk antibody (Immunoglobulin of yolk, IgY).Hen immunity can produce corresponding chicken yolk immune ball in latter 15 days
Albumen, and comparatively fast peaking, produces the immunne response of long period, and titer can maintain the several months to more than 1 year, and IgY contains
IgG in amount even more than chicken serum.The research of yolk antibody started from for 19 end of the centurys to 20 beginnings of the century.1893, Klemperer
The immunity demonstrating tetanus toxin can pass to chicken from hen, and in birds, the acquired of this passive immunity is just since then
Known to common people.Willianms etc. find that Yolk immunoglobulin is primarily present in the yolk of birds for 1962, and research finds,
P. aeruginosa verticillium toxin with inactivation or the Pseudomonas aeruginosa bacterial chip through ultrasonic Treatment are made antigen immune and are laid eggs
After chicken, the Pseudomonas aeruginosa that burnt degree is caused by isolated resisting pseudomonas aeruginosa Yolk immunoglobulin from its yolk
Infection has significant preventive and therapeutic action.Due to this antibody rich content in Ovum Gallus domesticus Flavus, and utilize specific antigen repeatedly
Laying hen after immunity can produce corresponding specificity high immunity yolk antibody.Since the eighties in 20th century, IgY antibody starts extensively should
With, yolk antibody is a great discovery of current bioscience research field.Along with laboratory and clinical research antagonist need
The increase asked, cheap extensive antibody production techniques has become new research topic, utilizes that super to exempt from chicken stable by specific antibody
Reaching constantly in yolk, prepare specific polyclonal antibody, low cost and yield are high, are expected to the scale that becomes and prepare specificity and resist
The effective means of body.
That IgY separates it is crucial that remove fat and the lipoprotein of Gao Cangliang in yolk, it is thus achieved that water-soluble component (Water
Soluble Fraction WSF).A very long time, owing to being difficult to be isolated from abundant yolk lipid by IgY antibody
Come so that egg is restricted as the research of antibody sources.
1) polyethylene glycol precipitation
First Polyethylene Glycol (PEG) sedimentation method are proposed by Poison.PEG is non-ionic water-soluble polymer, high concentration
Not causing protein denaturation, the sedimentation time is shorter than sulphuric acid money and ethanol, does not affects centrifugal treating.Precipitate with 12%PEG6000
WSF can be obtained with 10000r/min low-temperature centrifugation 25min.This method antibody response rate is high, and antibody activity is not destroyed, but this method
Operating procedure is numerous and diverse, can not remove lipid after being centrifuged completely, and Polyethylene Glycol residual is not readily removable.
2) water dilution method
Use distilled water diluting yolk, with the pH of diluted acid (hydrochloric acid, octanoic acid etc.) regulation distilled water, receive after lipoprotein natural sedimentation
Collection supernatant i.e. obtains WSF.Dilute with water method goes fat to be to go the best approach in fat research at present, and it is without chemical reagent, no
Damage IgY activity, safety is good, and do not affect lipid further with.
3) organic solvent method
Utilizing the fatsolvent such as chloroform, ether to be sufficiently mixed with the yolk solution of normal saline dilution, lipoprotein is denatured solidifying
Knot, removes the organic solvent of lower floor and the denatured lipoprotein in intermediate layer the most afterwards, collects supernatant and is purification IgY.This method
Remove lipid ratio more thoroughly, supernatant clear, the antibody response rate is high, but residual organic solvent in supernatant, antagonist is lived
Property has an impact, and the environmental pollution ratio that organic solvent causes simultaneously is more serious, thus this method is suitable only for laboratory and prepares on a small quantity.
Summary of the invention
It is an object of the present invention to provide a kind of canine distemper-infectious hepatitis-parvovirus Triple egg yolk antibody.
Another object of the present invention is to provide described canine distemper-infectious hepatitis-parvovirus Triple egg yolk antibody
Preparation method.
For reaching above-mentioned purpose, on the one hand, the invention provides a kind of canine distemper-infectious hepatitis-parvovirus three ovum
The preparation method of yellow antibody, wherein, described method includes:
(1) immunity: by 25 week old ISA laying hens raise 7-10 days, with containing canine distemper virus, canine infectious hepatitis virus,
The commercially available polyvalent vaccine immunity of Canine Parvovirus laying hen after raising, 1.5-2 plumage part/only, collect egg, measure antibody and drip
Degree, if antibody titer not up to requires level, again immunity, and continues to collect egg, measures antibody titer, until titre reaches
To requiring level, and determine immune time.
(2) extract: the titre taking step (1) reaches the egg of requirement level, surface sterilization, separates egg yolk, homogenate, then
Dilute with sterile distilled water, regulate pH to 5.0-5.2, after standing, collect supernatant；Supernatant acetate buffer dilutes, and adjusts
Joint pH to 5.0-5.2, adds caprylic acid, stratification, and water intaking is centrifuged mutually, takes precipitation and is dissolved in normal saline, is filtrated to get described ovum
Yellow antibody.
According to some specific embodiments of the present invention, wherein, step (1) is to collect egg 10-15 days continuously, measures antibody
Titre.
According to some specific embodiments of the present invention, wherein, step (2) described surface sterilization includes putting into egg the cleanest
You go out and soak in solution, and taking-up is dried, then processed eggshell surface with ethanol water.
According to some specific embodiments of the present invention, wherein, preferably egg is put into immersion 10-in bromogeramine solution
30min。
According to some specific embodiments of the present invention, wherein, further preferably bromogeramine solution mass concentration is 0.5%.
According to some specific embodiments of the present invention, wherein, further preferably with ethanol water, eggshell surface is processed
For eggshell surface being carried out wiping with ethanol water.
According to some specific embodiments of the present invention, wherein, further preferably the mass concentration of ethanol water is 70-80%.
According to some specific embodiments of the present invention, wherein, step (2) is to use sterile distilled water with sterile distilled water dilution
It is diluted to 8-12 times of the volume after egg yolk homogenate；Wherein it is preferably 10 times.
According to some specific embodiments of the present invention, wherein, after step (2) regulation pH to 5.0-5.2, it is at 2-10 DEG C
Under, stand 6h-10h, regather supernatant；Wherein it is preferably at 4 DEG C, stands 6h.
According to some specific embodiments of the present invention, wherein, the acetate buffer of step (2) be pH be 5.0,
The acetate buffer of 0.1mol/L.
According to some specific embodiments of the present invention, wherein, step (2) is dilution 2 times with acetate buffer dilution.
According to some specific embodiments of the present invention, wherein, step (2) adds caprylic acid to concentration is 1%.
According to some specific embodiments of the present invention, wherein, step (2) is centrifugal is under 12000rpm rotating speed, at 4 DEG C
Centrifugal 30min.
According to some specific embodiments of the present invention, wherein, step (2) is filtrated to get described yolk with 0.22 μm filter
Antibody.
According to some specific embodiments of the present invention, wherein, step (2), after separating egg yolk, first removes egg yolk outer layer
Film, then egg yolk is homogenized.
According to some specific embodiments of the present invention, wherein, method of the present invention includes:
The immunity of 1 laying hen
Being raised precedently by the laying hen of purchase one week, the feedstuff fed is all consistent with before purchase with light application time length so that it is suitable
Should new environment, minimizing stress.
With canine distemper-infectious canine hepatitis-canine parvovirus disease 3 vaccines immunity test group chicken, 2 plumage parts/only.Immunity
After, collect egg 2 weeks continuously, measure antibody titer.If antibody titer not up to requires level, booster immunization, collect egg, survey
Determine antibody titer, till reaching titre requirement level, and determine immune time.
The separation and Extraction of 2 yolk antibodies
First egg tap water is rinsed well, then egg is put into immersion 20min in 0.5% bromogeramine solution
After, taking-up is dried.With 75% alcohol swab wiping eggshell, sterile working opens eggshell, Ovum Gallus domesticus album and egg yolk is separated, takes egg yolk and be put in
In sterilizing beaker, the careful film removing egg yolk, it is sufficiently stirred for beating, makes homogenate.Yolk liquid sterile distilled water is diluted 10
Times, adjustment pH value is between 5.0～5.2, and 4 DEG C stand 6h, collect supernatant.Supernatant with acetate buffer (pH5.0,
0.1mol/L) diluting 2 times, adjust pH value to 5.0, add caprylic acid extremely final concentration of 1%, stratification, yolk antibody is in water layer.
Water intaking phase, 12000r/min, 4 DEG C, 30min is centrifuged, and takes precipitation and is dissolved in a small amount of normal saline, crosses with 0.22 μm filter and filter
Bacterium, obtains yolk antibody IgY, 4 DEG C of preservations.
On the other hand, present invention also offers canine distemper-infectious hepatitis that described preparation method prepares-tiny
Virus Triple egg yolk antibody.
In sum, the invention provides a kind of canine distemper-infectious hepatitis-parvovirus Triple egg yolk antibody and system thereof
Standby.The yolk antibody of the present invention has the advantage that
1. immunizing antigen is the most remote with the species sibship of body, the easiest by body immune system identification.Due to chicken with
Farther out, therefore can produce for dog is easy by the conservative protein antibody of body immune system identification for the phylogenesis of dog.
2. the present invention is directed to canine distemper, yolk antibody that infectious canine hepatitis, Canine Parvovirus are prepared from not with class wind
The wet factor (RF) and dog complement combine, and decrease the interference of immune detection, improve accuracy.
3. after specific antigen immunity, the persistency response that hen creates antagonism former, can constantly obtain specific many grams
Grand antibody, it derives from same individuality, and antibody homogeneity is good.
3. the yolk antibody content in yolk is apparently higher than the content of lgG in chicken serum, about 15-25mg/mL yolk.?
In identical immunization time, the amount of antibody extracted in a chicken institute raw egg it is 120 times prepared by the serum of a rabbit.
4. yolk antibody can resist the gastric acid barrier action of young dog, and can trypsin and chymotrypsin protein in anti-intestinal
The digestion of enzyme.
5. the stable in physicochemical property of yolk antibody, convenient sources, yield is high, low cost, and animal germline genetis method away from
From advantage, be more suitable for produce specific antibody, may be used for preparing diagnostic reagent, carrying out the preventing and treating of disease and add as feedstuff
Add agent, there is the potential of development functionality food and new drug.
Detailed description of the invention
Implementation process and the beneficial effect of generation of the present invention is described in detail, it is intended to help to read below by way of specific embodiment
Reader is more fully understood that essence and the feature of the present invention, not as can the restriction of practical range to this case.
Embodiment 1
The immunity of 1 laying hen
By buy 35 week old ISA laying hens be raised precedently 7-10 days, the feedstuff fed and light application time length all with purchase before
Unanimously so that it is shake down, minimizing stress.
120 laying hens are randomly divided into 2 groups: A group 40 (test group 1), and B group 40 (tests 2 groups), C group 40 (comparison
Group).
A group: with the polyvalent vaccine immunity A group chicken containing canine distemper-infectious canine hepatitis-canine parvovirus disease, 1.5 plumages
Part/only.After immunity, collect egg 2 weeks continuously, measure antibody titer.Whether antibody titer reaches requirement level (1: 512), if
Antibody titer not up to requires level, booster immunization, collects egg, measures antibody titer, till reaching titre requirement level, and
Determine immune time.
B group: with the polyvalent vaccine immunity B group chicken containing canine distemper-infectious canine hepatitis-canine parvovirus disease, 2.0 plumages
Part/only.After immunity, collect egg 2 weeks continuously, measure antibody titer.Whether antibody titer reaches requirement level (1: 512), if
Antibody titer not up to requires level, booster immunization, collects egg, measures antibody titer, till reaching titre requirement level, and
Determine immune time.
B group: immunization method is all consistent with A group with immunization time, every time injecting normal saline during immunity.
The separation and Extraction of 2 yolk antibodies
First egg tap water is rinsed well, then egg is put into immersion 10-in 0.5% bromogeramine solution
After 30min, taking-up is dried.With 75% alcohol swab wiping eggshell, sterile working opens eggshell, Ovum Gallus domesticus album and egg yolk is separated, takes egg
Huang is put in sterilizing beaker, the careful film removing egg yolk, is sufficiently stirred for beating, makes homogenate.By yolk liquid sterile distilled water
Diluting 10 times, adjustment pH value is between 5.0～5.2, and 4 DEG C stand 6h, collect supernatant.Supernatant acetate buffer
(pH5.0,0.1mol/L) dilutes 2 times, adjust pH value to 5.0, add caprylic acid to its final concentration of 1%, stratification, yolk antibody
In water layer.Water intaking phase, 12000r/min, 4 DEG C, 30min is centrifuged, and takes precipitation and is dissolved in a small amount of normal saline, by 0.22 μm mistake
Filter filtration sterilization, obtains yolk antibody IgY, 4 DEG C of preservations.
Embodiment 2
The preparation of canine distemper-infectious canine hepatitis-canine parvovirus disease 3 egg yolk antibody injection
First, by the normal saline solution reaching certain standard yolk antibody preparing antibody titer, wheat is added
Bud sugar (90-100g/L), as stabilizer, adds thimerosal (0.003%～0101%) and, as preservative, joins with water for injection
Put, then by coarse filtration, fine straining, fill, sterilizing, lamp inspection, labeling, pack, put in storage, obtain canine distemper-infectious canine hepatitis-dog thin
Minor illness viral disease 3 egg yolk antibody injection finished product.
Test case 1
The titration of yolk antibody
1, neutralization test
(1) CDV, CAV, CPV virus stock solution used having measured poison valency in advance is diluted to 200TCID50/0.1mL。
(2) the yolk antibody normal saline dilution that will separate so that it is dilution factor is respectively 1: 2,1: 4,1: 8,1: 16,1:
32、1∶64、1∶128、1∶256、1∶512、1∶1024。
(3) take the virus liquid 0.5mL diluted, mix from different dilution antibody equivalent, 37 DEG C of water-bath effect 1h.Often
One dilution factor is inoculated 3～6 bottles of cells and is cultivated (Vero cell), observation of cell pathological changes.The virus liquid pair being not added with antibody is set simultaneously
According to and blank, arrange if desired monoclonal antibody comparison.
(4) statistics cytopathy parameter, occurs without cytopathic antibody maximum dilution multiple, is antibody titer.
2, Elisa measures canine distemper antibody titer
Buy canine distemper virus antibody (CDV-Ab) ELISA detection kit, detect yolk antibody by test kit description
Middle canine distemper virus antibody titer titre.
3, blood clotting and hemagglutination inhibition test measure infectious canine hepatitis antibody titer
Buy canine infectious hepatitis virus antibody (ICHV-Ab/CAV-Ab) blood clotting and blood clotting suppression detection kit, normally
Canine infectious hepatitis virus antibody titer titre in bright book detection yolk antibody.
4, blood clotting and hemagglutination inhibition test measure canine parvovirus disease antibody titer
Buy canine parvovirus disease antibody (CPV-Ab) blood clotting and blood clotting suppression detection kit, by specification detection yolk
Canine Parvovirus antibody titer titre in antibody.
5, antibody titer testing result
Result shows, A group and B group test chicken all after the 3rd immunity, canine distemper-infectious canine hepatitis-Canine Parvovirus
3 kinds of antiviral antibody titres all reach peak demand level (1: 512), and the egg antibody titer after the 3rd immunity 20 days starts
Decline, less than peak demand level.Therefore, 35 week old ISA laying hens immunity contain canine distemper-infectious canine hepatitis-Canine Parvovirus
Viral vaccine 1.5-2 plumage part in 3/only, every immunity in 15 days once, immunity 3 times altogether, after the 3rd immunity, antibody titer reaches
High level (1: 512).
Test case 2
The controlling experiment of 1 egg yolk antibody injection
The preventing and treating to canine distemper disease of 1.1 egg yolk antibody injection
1) prophylactic tria
Selection average weight is about 5kg, clinical examination healthy, and body temperature is normal, and change of serum C DV quick detection test paper detects
The common pup 15 at 3～4 monthly ages that CDV is negative, is randomly divided into three groups of I groups (matched group), II group (yolk antibody group), III
(monoclonal antibody group).I group is conventional raises, and II group is on the basis of routine is raised, and according to dosage intramuscular injection yolk antibody is injected
Liquid, every day 1 time, continuous 3 days, III group routine raise on the basis of, according to dosage intramuscular injection monoclonal antibody, every day 1 time,
Continuous 3 days.All test dogs on the 4th start, with CDV poison tissue fluid, oral administration 2ml, subcutaneous injection 2ml by force, to carry out counteracting toxic substances, see
Examine its incidence.Criterion: spirit is depressed, loss of appetite, body temperature raises, and eye, nose flow out mucus or purulent secretion,
Asoscope is dry and cracked, vomiting, and diarrhoea waits, CDV reagent paper Virus monitory positive is judged to morbidity.The yolk antibody prevention to canine distemper virus
Result of the test (being shown in Table 1) shows, I group sickness rate is 100%, and the sickness rate of II group and III group is 60%.II group and III group
Sickness rate significantly lower than I group.Result shows, similar to monoclonal antibody, and the yolk antibody of this research has preventing canine distemper
Effect.
Table 1 yolk antibody prophylactic tria result to canine distemper virus
2) therapeutic test
Select the morbidity pup 20 of natural infection canine distemper clinically, by canine distemper virus (CDV) detection paper be all
Strong positive, and the classical symptom of canine distemper occurs: spirit is depressed, loss of appetite, body temperature raises, eye, nose flow out mucus or
Purulent secretion, asoscope is dry and cracked, vomiting, diarrhoea etc..
Ill dog is divided into two groups: A group (test group) 10, B group (matched group) 10.A group according to dosage intramuscular injection ovum
Yellow antibody is treated, and B group is according to dosage injected canine distemper virus monoclonal antibody and treated, and finally adds up the healing of ill dog
Rate, average cure time.Yolk antibody can effectively treat canine distemper (the results are shown in Table 2).
Table 2 yolk antibody treatment situation to canine distemper
After treatment after a while, test group has 4 dogs to cure, 7 dog death, average cure time 12 days.Right
2 dogs are had to cure according to group, 8 dog death, average cure time 17 days.
The preventing and treating to infectious canine hepatitis of 2 egg yolk antibody injection
1) prophylactic tria
Selection average weight is about 5kg, clinical examination healthy, and body temperature is normal, and whole blood ICHV quick detection test paper detects
The common pup 15 at 3～4 monthly ages that ICHV is negative, is randomly divided into three groups of I groups (matched group), II group (yolk antibody group), III
(ribavirin group).I group is conventional raises, II group on the basis of routine is raised, according to dosage intramuscular injection egg yolk antibody injection,
Every day 1 time, continuous 3 days, III group routine raise on the basis of, according to dosage intramuscular injection ribavirin, every day 1 time, continuous 3
Day.All test dogs on the 4th start, with ICHB poison tissue fluid, oral administration 2ml, subcutaneous injection 2ml by force, to carry out counteracting toxic substances, observe it
Incidence.Criterion: vomit, suffer from abdominal pain, suffer from diarrhoea, body temperature rises high, ICHV reagent paper positive blood tests person and is judged to morbidity.Ovum
The prophylactic tria result (being shown in Table 3) of canine infectious hepatitis virus is shown by yellow antibody, and I group sickness rate is 100%, II group and III
The sickness rate of group is 80%.The sickness rate of II group and III group is substantially less than I group.Result shows, and when ribavirin preventing canine
The effect of infectious hepatitis is similar, and the yolk antibody of this research has the effect of preventing canine infectious hepatitis.
Table 3 yolk antibody prophylactic tria result to canine infectious hepatitis virus
2) therapeutic test
Select the morbidity pup 20 of natural infection infectious canine hepatitis clinically, with infectious canine hepatitis (ICHV) reagent paper
Detection is all strong positive, and the classical symptom of canine parvovirus disease occurs: vomit, suffer from abdominal pain, suffer from diarrhoea, body temperature rises high.
Ill dog is divided into two groups: A group (test group) 10, B group (matched group) 10.A group according to dosage intramuscular injection ovum
Yellow antibody is treated, and B group is according to dosage injected ribavirin and treated, and finally adds up the cure rate of ill dog, averagely cures
Time.Result shows, yolk antibody can effectively treat infectious canine hepatitis (the results are shown in Table 4).
Table 4 yolk antibody treatment situation to infectious canine hepatitis
After there is disease, A, B two groups gives emergency treatment, and test group is cured by 8 dogs, and average cure time is 5 days；Comparison
Organizing 6 dogs to cure, average cure time is 7 days.Other dog is all dead within morbidity 0.5-2 days, and occur vomiting, diarrhoea,
Stomachache, body temperature rise high symptom.
The preventing and treating to canine parvovirus disease of 3 egg yolk antibody injection
1) prophylactic tria
Selection average weight is about 5kg, clinical examination healthy, and body temperature is normal, and feces CPV quick detection test paper detects
The common pup 20 at 3～4 monthly ages that CPV is negative, is randomly divided into 2 groups, I group (yolk antibody group), II (monoclonal antibody group).
I group is conventional raises, II group on the basis of routine is raised, according to dosage intramuscular injection egg yolk antibody injection, every day 1 time, continuous 3
Day, III group routine raise on the basis of, according to dosage intramuscular injection monoclonal antibody, every day 1 time, continuous 3 days.Within 4th, own
Test dog starts, with CPV poison tissue fluid, oral administration 2ml, subcutaneous injection 2ml by force, to carry out counteracting toxic substances, observe its incidence, wherein
Criterion: pup occurs that vomiting, diarrhoea or hemafecia, feces rise high, CPV reagent paper feces detection with off-odor, body temperature
Positive is judged to morbidity.The prophylactic tria result (being shown in Table 5) of Canine Parvovirus is shown by yolk antibody, and I group sickness rate is
The sickness rate of 100%, II group and III group is 60%.The sickness rate of II group and III group is significantly lower than I group.Result shows, with
Monoclonal antibody is similar, and the yolk antibody of this research has the effect of preventing canine parvovirus infection.
Table 5 yolk antibody prophylactic tria result to Canine Parvovirus
2) therapeutic test
Select the morbidity pup 20 of natural infection canine parvovirus disease clinically, examine with Canine Parvovirus (CPV) reagent paper
Surveying is all strong positive, and the classical symptom of canine parvovirus disease occurs: pup occur vomiting, diarrhoea or hemafecia, feces with
Off-odor, body temperature rise high.
Ill dog is divided into two groups: A group (test group) 10, B group (matched group) 10.A group according to dosage intramuscular injection ovum
Yellow antibody is treated, and B group is according to dosage injected Canine Parvovirus monoclonal antibody and treated, and finally adds up the healing of ill dog
Rate, average cure time.Result shows, yolk antibody can effectively treat canine parvovirus disease (the results are shown in Table 6).
Table 6 yolk antibody treatment situation to canine parvovirus
After treatment, test group has 1 dog at the 7th day because secondary myocarditis is dead, and 9 dogs are cured, and averagely cure
Time is 4 days.
Matched group has 3 dog death, and wherein 2 dogs cause cylinder electrolyte disorder, acidosis at the 4th day and 5 because being dehydrated
It is dead, and a dog is dead because of secondary myocarditis at the 8th day；Having 7 dogs to cure, average cure time is 6 days.
Claims (10)
1. a preparation method for canine distemper-infectious hepatitis-parvovirus Triple egg yolk antibody, wherein, described method includes:
(1) immunity: raised 7-10 days by 25 week old ISA laying hens, with thin containing canine distemper virus, canine infectious hepatitis virus, dog
The commercially available polyvalent vaccine immunity of small virus laying hen after raising, 1.5-2 plumage part/only, collect egg, measure antibody titer,
If antibody titer not up to requires level (1: 512), again immunity, and continues to collect egg, measure antibody titer, until dripping
Degree reaches requirement level, and determines that immune time is 3 times, collects egg 10-15 days reaching antibody titer levels；
(2) extract: the titre taking step (1) reaches the egg of requirement level, surface sterilization, separates egg yolk, homogenate, use aseptic steaming
Distilled water dilutes, and regulates pH to 5.0-5.2, collects supernatant after standing；Supernatant acetate buffer dilutes, and regulation pH is extremely
5.0-5.2, adds caprylic acid, stratification, and water intaking is centrifuged mutually, takes precipitation and is dissolved in normal saline, is filtrated to get described yolk antibody；
The most centrifugal is under 12000rpm-13000rpm rotating speed, centrifugal 30-40min at 4 DEG C；
Further preferably it is filtrated to get described yolk antibody with 0.22 μm filter.
Preparation method the most according to claim 1, wherein, step (1) is to collect egg 10-15 days continuously, randomly draws
18-20 piece of egg, measures antibody titer.
Preparation method the most according to claim 1, wherein, step (2) described surface sterilization includes putting into egg the cleanest
You go out and soak in solution, and taking-up is dried, then processed eggshell surface with ethanol water；The most preferably egg is put into newly
Geramine solution soaks 10-30min；The most further preferably bromogeramine solution mass concentration is 0.5%；The most further preferably use second
Eggshell surface is processed as, with ethanol water, eggshell surface is carried out wiping by alcohol-water solution；The most further preferably ethanol is water-soluble
The mass concentration of liquid is 70-80%.
Preparation method the most according to claim 1, wherein, step (2) is to use aseptic steaming with sterile distilled water dilution (V: V)
To 8-12 times of volume after distilled water dilution egg yolk homogenate；More preferably 10 times.
Preparation method the most according to claim 1, wherein, after step (2) regulation pH to 5.0-5.2, is at 2-10 DEG C
Stand 6h-10h, regather supernatant；Wherein it is preferably at 4 DEG C, stand 6h.
Preparation method the most according to claim 1, wherein, the acetate buffer pH of step (2) is 5.0,0.1mol/L
Acetate buffer.
Preparation method the most according to claim 1, wherein, step (2) is dilution 2 with acetate buffer dilution (V: V)
Times.
Preparation method the most according to claim 1, wherein, step (2) add caprylic acid to its final concentration of 1%-2% (V:
V)。
9., according to the preparation method described in claim 1～8 any one, wherein, step (2), after separating egg yolk, first removes
The film of egg yolk outer layer, then egg yolk is homogenized.
10. canine distemper-infectious hepatitis-parvovirus that the preparation method described in claim 1～9 any one prepares
Triple egg yolk antibody.
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