CN102735807A - Effect testing method for duck hemorrhagic ovaritis inactivated vaccine - Google Patents
Effect testing method for duck hemorrhagic ovaritis inactivated vaccine Download PDFInfo
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Abstract
The present invention provides a serology effect testing method for a duck hemorrhagic ovaritis inactivated vaccine. According to the method, a duck hemorrhagic ovaritis inactivated vaccine is adopted to vaccinate ducks twice, the second time vaccination is performed after the first time vaccination is performed two weeks, duck serums are sampled to carry out ELISA antibody detection after the second time vaccination is performed 3-5 weeks, wherein the qualified standard of the vaccine effect test is that the ELISA antibody positive rate of the non-vaccinated group is 0% and the ELISA antibody positive rate of the vaccinated group is not less than 80%. The serology effect testing method for the duck hemorrhagic ovaritis inactivated vaccine in the present invention has characteristics of convenient operation and accurate result, and can be widely used for effect evaluations of duck hemorrhagic ovaritis inactivated vaccines.
Description
Technical field
The present invention relates to a kind of method for testing efficacy of inactivated vaccine for animals, relate in particular to the method for inspection that a kind of duck hemorrhagic oaritis inactivated vaccine is renderd a service, belong to veterinary biologics inspection technology field.
Background technology
Duck hemorrhagic oaritis (Duck Hemorrhagic Ovaritis; DHO) be a kind of novel duck infectious disease popular recently, acute, the height contact, its cause of disease be duck hemorrhagic oaritis virus (Duck Hemorrhagic Ovaritis Virus, DHOV); The member who on virus taxis, belongs to flaviviridae; Infect the different days duck, cause that the ovarian hemorrhage of sexal maturity duck, orchiatrophy, egg production, rate of fertilization and incubation rate descend, the duck death rate increases.
In the research of virus taxis status; Through the F4 of isolated viral strain is carried out sequence homology analysis and evolutionary analysis for the go down to posterity genome sequence of virus and known virus of duck embryo, find that the genetic distance of tembusu virus of this virus and flaviviridae Flavivirus is nearest.Aspect diagnosis, Cao Zhenzhen, Wan Chun and, Su Jingliang, Teng Qiao are vast etc., and people's report can adopt clinical symptoms, reach methods such as histopathology changes, the RT-PCR amplification separates with virus substantially makes a definite diagnosis this disease.About these sick prevention and control, the strain that Beijing City Agriculture and Forestry Institute and Ruipu (Baoding) Biological Pharmaceutical Co., Ltd., Qilu Animal Health Products Co., Ltd., Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute and academy of agricultural sciences, Chongqing City animal and veterinary research institute have been separated during having reported and having utilized this disease to break out prepares the method for vaccine.Lin Jian, Liu Yuehuan etc. have reported the artificial challenge's model that utilizes laying ducks successfully to set up duck hemorrhagic oaritis, for experiment basis has been established in the evaluation of vaccine.That Shang Weijian has is objective, feasible, the play-by-play of the vaccine potency evaluation method of strong operability and science.
Summary of the invention
One of the object of the invention provides the age in days scope of a kind of duck hemorrhagic oaritis inactivated vaccine effect evaluation duck.
Two of the object of the invention provides the tissue samples of duck hemorrhagic oaritis inactivated vaccine effect evaluation with vaccine immunity number of times, sampling time, collection.
Three of the object of the invention provides a kind of duck hemorrhagic oaritis inactivated vaccine effect evaluation method.
The used virus stain called after of the present invention duck hemorrhagic oaritis virus HB strain, preserving number is CCTCC V201122; Said duck hemorrhagic oaritis virus is ssRNA virus, and cyst membrane is arranged, can aggegation dove red blood cell; It is spherical that virion is substantially, diameter 40～60nm; The said virus resistibility of environment to external world is stronger, in 4 ℃, deposits several weeks, in-20 ℃, deposits some months, and its appeal is all unaffected; Said virus is responsive to ether, chloroform; Most of detergents can be with its rapid deactivation; Under 37 ℃ of conditions, stifling 6h just can be its deactivation with 0.1% formalin.This virus can be at 9～13 age in days duck embryo allantoic cavities, embryo CAM, 9～10 age in days chick embryo allantoic cavities, embryo CAM, and 6 age in days chick embryo yolk sacs are cultivated in 7～8 age in days duck embryo yolk bags, also can on DEF and BHK21 cell, cultivate.
The preparation method of the used duck hemorrhagic of the present invention oaritis inactivated vaccine has declared patent of invention in 2011, application number is 201110255115.1.
The present invention provides a kind of duck hemorrhagic oaritis inactivated vaccine method for testing efficacy; This method is used duck hemorrhagic oaritis inactivated vaccine immune duck; Immunity is 2 times altogether, carries out 22 weeks after head exempts from and exempts from, and the duck serum that 3 ~ 5 weeks of back are exempted from collection two carries out the ELISA antibody test; Non-immune control group duck ELISA antibody positive rate is 0%, and the ELISA antibody positive rate is judged to vaccine potency and is up to the standards for being not less than 80%.
Further, the inventive method confirms that according to the neurological susceptibility experimental result it is 42 ~ 560 ages in days that duck hemorrhagic oaritis inactivated vaccine efficacy test uses the age in days of duck.
Further, in the inventive method the dosage of 2 immunity be 0.5ml/ only～2ml/ only.
Further; A kind of duck hemorrhagic oaritis inactivated vaccine method for testing efficacy provided by the invention is to use duck hemorrhagic oaritis inactivated vaccine immunity 42～560 age in days ducks; Carrying out 22 weeks after head exempts from exempts from; Gather two and exempt from the back duck serum in 3 ~ 5 weeks, use the ELISA antibody assay kit to detect immune duck serum, carry out effect evaluation.
Further, above-mentioned ELISA antibody test is that the flaviviridae with purifying is the ELISA detection kit of envelope antigen.
Further, described duck is that meat duck, egg are with duck or SPF duck.
A kind of duck hemorrhagic oaritis inactivated vaccine method for testing efficacy provided by the invention specifically is to use duck hemorrhagic oaritis inactivated vaccine immunity 42～560 age in days ducks, establishes the non-immune control group duck of some simultaneously; Carrying out 22 weeks after head exempts from exempts from; The dosage of twice immunity only is 0.5ml/～2ml/, gathers two and exempts from the back duck serum in 3 ~ 5 weeks, and use ELISA antibody assay kit detects immune duck serum; Carry out effect evaluation; Non-immune control group duck ELISA antibody positive rate is 0%, and immune group duck ELISA antibody positive rate >=80% judges that vaccine potency is up to the standards.
Further, above-mentioned ELISA antibody assay kit is that the flaviviridae with purifying is the ELISA detection kit of envelope antigen.
Further, described duck is that meat duck, egg are with duck or SPF duck.
The tissue samples of the immune time of vaccine, the time of drawing materials, collection had proposed the qualified standard of serology ELISA vaccine potency test method, criterion and vaccine quality when the present invention had proposed to be used for age in days that duck hemorrhagic oaritis inactivated vaccine serology efficacy test uses duck, vaccine potency check first.Solved the blank of duck hemorrhagic oaritis inactivated vaccine serology effect evaluation method and criterion.The method that the present invention confirms is objective, comprehensive, strong operability, can be used for the laboratory and the clinical efficacy evaluation of duck hemorrhagic oaritis inactivated vaccine.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, it is commercially available that used biology, chemical reagent is routine among the embodiment, the conventional means that used technological means is well known to those skilled in the art among the embodiment.Beijing duck used among the embodiment is available from duck hemorrhagic oaritis negative antibody duck field (Nan Kou of Beijing Golden Star Duck Center kind duck field); Sheldrake is available from duck hemorrhagic oaritis negative antibody duck field (dress circle village under four seas town, Yanqing County, Beijing Pearl Spring); The SPF duck is raised to testing required age in days in the SPF chicken isolator by the SPF duck's egg hatching available from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
The test stone of susceptible duck
5 from without duck hemorrhagic oaritis inactivated vaccine immunity or the popular history duck of duck hemorrhagic oaritis crowd's duck is arranged, and strain is used in the check of 100 times of dilutions of every oral 1.0 milliliters of aseptic 0.5% lactoalbumin hydrolysate Hank ' s liquid of duck.Attack poison back 4-5 day, blood sampling, separation of serum carries out virus to be separated.The virus separation rate should at least 4/5 positive, judges that check uses duck to be the susceptible duck.
Embodiment 1 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) efficacy test is with age in days, immunizing dose, the sampling time (1) of duck
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201101; The development of Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu (Baoding) Biological Pharmaceutical Co., Ltd.) immune 560 ages in days and do not infect 10 of the sheldrakes of DHOV; 0.5ml/ only, chest muscle injection.2 weeks carrying out two according to same dose and identical approach after head exempts from exempts from; The serum of 4 all ducks behind the collection secondary immunity; Use the ELISA antibody detection method of note to detect immune duck serum; Non-immune control group duck ELISA antibody positive rate is 0%, and immune group duck ELISA antibody positive rate is 80%, explains that vaccine potency is up to the standards.
Embodiment 2 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) efficacy tests are with age in days, immunizing dose, the sampling time (2) of duck
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201102, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 42 ages in days and do not infect 10 of the Beijing ducks of DHOV, 1ml/ only, chest muscle is injected.Head exempts from the back and exempted from identical approach two according to same dose in 2 weeks; The serum of 4 all ducks behind the collection secondary immunity; Use the ELISA antibody detection method of note to detect immune duck serum; Non-immune control group duck ELISA antibody positive rate is 0%, and immune group duck ELISA antibody positive rate is 90%, explains that vaccine potency is up to the standards.
Embodiment 3 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) efficacy tests are with age in days, immunizing dose, the sampling time (3) of duck
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201103; The development of Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu (Baoding) Biological Pharmaceutical Co., Ltd.) immune 180 ages in days and do not infect 10 of the Beijing ducks of DHOV; 2ml/, the chest muscle injection.Head exempts from the back and exempted from identical approach two according to same dose in 2 weeks; The serum of 4 all ducks behind the collection secondary immunity; Use the ELISA antibody detection method of note to detect immune duck serum; Non-immune control group duck ELISA antibody positive rate is 0%, and immune group duck ELISA antibody positive rate is 100%, explains that vaccine potency is up to the standards.
Embodiment 4 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) efficacy tests are with age in days, immunizing dose, the sampling time (4) of duck
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201104; The development of Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu (Baoding) Biological Pharmaceutical Co., Ltd.) immune 560 ages in days and do not infect 10 of the SPF ducks of DHOV; 0.5ml/ only, chest muscle injection.2 weeks carrying out two according to same dose and identical approach after head exempts from exempts from; The serum of 3 all ducks behind the collection secondary immunity; Use the ELISA antibody detection method of note to detect immune duck serum; Non-immune control group duck ELISA antibody positive rate is 0%, and immune group duck ELISA antibody positive rate is 80%, explains that vaccine potency is up to the standards.
Embodiment 5 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) efficacy tests are with age in days, immunizing dose, the sampling time (5) of duck
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201105, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 300 ages in days and do not infect 10 of the sheldrakes of DHOV, 1ml/ only, chest muscle is injected.2 weeks carrying out two according to same dose and identical approach after head exempts from exempts from; The serum of 5 all ducks behind the collection secondary immunity; Use the ELISA antibody detection method of note to detect immune duck serum; Non-immune control group duck ELISA antibody positive rate is 0%, and immune group duck ELISA antibody positive rate is 80%, explains that vaccine potency is up to the standards.
Embodiment 6 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) efficacy tests are with age in days, immunizing dose, the sampling time (6) of duck
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201106; The development of Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu (Baoding) Biological Pharmaceutical Co., Ltd.) immune 400 ages in days and do not infect 10 of the Beijing ducks of DHOV; 2ml/, the chest muscle injection.2 weeks carrying out two according to same dose and identical approach after head exempts from exempts from; The serum of 5 all ducks behind the collection secondary immunity; Use the ELISA antibody detection method of note to detect immune duck serum; Non-immune control group duck ELISA antibody positive rate is 0%, and immune group duck ELISA antibody positive rate is 80%, explains that vaccine potency is up to the standards.
The preparation and the TPPA of embodiment 7 direct competitive-ELISA kit
1. kit is formed
2. material
2.1 biomaterial
The antigen purification viral antigen
Antibody antigen virus monoclonal antibody
Horseradish peroxidase
2.2 consumptive material
ELISA Plate Shenzhen bright China of gold
Container (bottle) Shenzhen bright China of gold
2.3ELISA plate preparation
2.3.1 the cell adapted Strain DHOV-HB strain of purifying duck hemorrhagic oaritis of virus (F5 generation) inoculation DEF (DEF), when CPE occurs 85%, results virus.After the freeze thawing 1 time, the centrifugal 15min of 3500rpm gets supernatant, again through the centrifugal 30min of 10000rpm; Get supernatant, concentrate, the PEG6000 deposition through hollow fiber column; SDGC is got the bright band between 30% and 45%, after dilution of STE damping fluid and centrifugal treating, obtains purified virus.
2.3.2 virus packets is utilized the carbonate buffer solution (prescription is seen note 1) of pH9.6 with 1000 times of dilutions of virus, joins in 96 orifice plates with the dosage in 100ul/ hole, encapsulates 12～16h under 4 ℃ of conditions.
Wash plate 5 times 2.3.3 wash the plate washing lotion, every hole is filled it up with.Each 30 seconds at interval.
2.3.4 ELISA Plate behind the plate is washed in sealing, claps and does.Add confining liquid 200ul.4 ℃ are spent the night, or 36 spend 1 hour.
2.3.5 dry sealing back ELISA Plate discards confining liquid, it is subsequent use to dry up the back.
2.3.6 Packaging Aluminum Foil encapsulation.
2.4 the preparation of enzyme labelled antibody employing sodium periodate method in duck hemorrhagic oaritis virus E protein monoclonal antibody, is prepared into the antibody that the ultraviolet spectrophotometer 280nm A of place value is about 2.5 enzyme conjugates marks with horseradish peroxidase-labeled.
2.5 the mensuration of sample antibody to be checked
2.5.1 add 50 μ L sample diluting liquids at each Kong Zhongjun of the elisa plate that has encapsulated;
2.5.2A1 hole and B1 hole respectively add 50 μ L positive serums; C1 hole and D1 hole respectively add 50 μ L negative serums, see preparation method (7); Add 50 μ L test serums in all the other each holes respectively.
2.5.321 ℃ ± 5 ℃ hatch 90min ± 6min.
2.5.4 remove each hole liquid, with 300 μ L washing fluids washing 3 times.
2.5.5 each hole adds the horseradish peroxidase-labeled antibody of 1 times of dilution of 100 μ L.
2.5.621 ℃ ± 5 ℃ hatch 30min ± 3min.
2.5.7 remove each hole liquid, with 300 μ L washing fluids washing 3 times.
2.5.8 add 100 μ L substrate solutions in each hole.
2.5.9 lucifuge is hatched 15min ± 2min for 21 ℃ ± 5 ℃.
2.5.10 in each hole, add 100 μ L stop buffer (2mol/L sulfuric acid) cessation reactions.
2.5.11 use ELIASA under the 450nm wavelength, to read and write down the OD value.
The result judges
The OD value of negative control is set up greater than 0.700 test.Calculate the S/N value of each sample: S/N=sample OD value/negative serum OD value * 100, and according to the form below is judged.
Points for attention
(1) substrate solution need keep in Dark Place, and does not contact with oxide.
(2) pollute for preventing, all used in test disposables need pass through autoclaving.
(3) 300 parts of negative serums of definite detection of critical value (cut-off) calculate its fiducial limit of 95%.Generally be constant of mean value X of negative control, this constant generally is to obtain (the normally negative mean value of 2.1X) according to 95% fiducial limit.
The prescription and the preparation of kit solution of the present invention or reagent
(1) pH9.6 encapsulates the prescription of damping fluid (CB)
(2) prescription of washing lotion
(3) prescription of sealing damping fluid
(4) prescription of sample diluting liquid or enzyme mark dilution
(5) stop buffer 2mol/L sulfuric acid.
(6) prescription of substrate solution A
The H of the prescription 30% of substrate solution B
2O
2
(7) preparation of positive and negative quality controlled serum is divided into 2 groups (infected group and non-infection control groups), 25 every group at random with 50 34 age in days SPF ducks.Oral administration (first) and intramuscular injection (secondary) route of inoculation infected duck hemorrhagic oaritis virus HB strain, infected duck and non-infection control group duck are all raised in SPF chicken isolator.Behind the superinfection 27 days, gather 2 groups of test duck blood, separation of serum filters, and every group of serum mixes, and adds thimerosal by the ratio of total amount 0.01%, and quantitative packing obtains 1 batch of positive serum and 1 batch of negative serum product.Positive serum and negative serum all do not have bacterial infection, and duck paramyxovirus, bird flu H5 hypotype, duck viral enteritis and duck hepatitis antibody are all negative, positive serum all can in 100 ELD
50Duck hemorrhagic oaritis virus, negative serum can not in F4 generation 100 ELD
50Duck hemorrhagic oaritis virus.
(8) preparation of DHOV-HB strain E protein monoclonal antibody designs a pair of primer (F5-GCGAATTCATCCTCCTGCTGTTGGTC-3 according to the primer of many strains flavivirus E gene of delivering on the GenBank; R5-ATCTGGAGTGACGCCCATCCAGAGT-3), adopt RT-PCR method amplification E gene order.The PCR product that reclaims is connected with pMD18-T, transforms the DH5a competent cell, conventional method is extracted and is identified plasmid, with positive plasmid called after pET-E.PET-E is expressed the E protein product that collection, evaluation and purifying are expressed in the Amp+LB fluid nutrient medium.With the E protein product of purifying with Freund's complete adjuvant and incomplete Freund emulsification, the preparation immunogene, immune BALB/c mouse when E albumen serum titer is qualified, is got spleen.Ratio according to 1:5 merges SP2/0 myeloma cell and immune spleen cell, and the positive hybridoma cell of screening and preparation secretion E protein antibodies is cloned positive hybridoma cell with limiting dilution assay.With the positive colony hybridoma and the paraffinum liquidum injection mouse peritoneal of above-mentioned some, after 2 weeks, gather the ascites of mouse, be the E protein monoclonal antibody.
Claims (8)
1. duck hemorrhagic oaritis inactivated vaccine method for testing efficacy; It is characterized in that, use duck hemorrhagic oaritis inactivated vaccine immune duck, immunity 2 times; Carrying out 22 weeks after head exempts from exempts from; Gather two exempt from 3 ~ 5 weeks of back the duck serum carry out the ELISA antibody test, non-immune group ELISA antibody positive rate is 0%, immune group ELISA antibody positive rate is judged to vaccine potency and is up to the standards for being not less than 80%.
2. the method for claim 1 is characterized in that, it is 42～560 ages in days that efficacy test uses the age in days of duck.
3. the method for claim 1 is characterized in that, the dosage of said 2 immunity be 0.5ml/ only～2ml/ only.
4. like the arbitrary said duck hemorrhagic oaritis inactivated vaccine method for testing efficacy of claim 1-3, it is characterized in that used ELISA antibody test is that flaviviridae with purifying is the ELISA detection kit of envelope antigen.
5. like the arbitrary said duck hemorrhagic oaritis inactivated vaccine method for testing efficacy of claim 1-3, it is characterized in that described duck is that meat duck, egg are with duck or SPF duck.
6. a duck hemorrhagic oaritis inactivated vaccine method for testing efficacy is characterized in that, uses duck hemorrhagic oaritis inactivated vaccine immunity 42～560 age in days ducks, carries out 22 weeks after head exempts from and exempts from, and the dosage of twice immunity only is 0.5ml/～2ml/; Gather two and exempt from the back duck serum in 3 ~ 5 weeks; Use the ELISA antibody assay kit to detect immune duck serum, carry out effect evaluation, non-immune control group duck ELISA antibody positive rate is 0%; Immune group duck ELISA antibody positive rate >=80% judges that vaccine potency is up to the standards.
7. method as claimed in claim 6 is characterized in that used ELISA antibody assay kit is that flaviviridae with purifying is the ELISA detection kit of envelope antigen.
8. like claim 6 or 7 described methods, it is characterized in that described duck is that meat duck, egg are with duck or SPF duck.
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| CN104977400A (en) * | 2015-07-27 | 2015-10-14 | 三明市和众生物技术有限公司 | Infertility joint detection kit and detection method thereof |
| CN105866424A (en) * | 2016-03-23 | 2016-08-17 | 北京市农林科学院 | Testing method for potency of inactivated vaccine against duck Tembusu viral diseases |
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| CN102735807B (en) | 2015-01-14 |
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