CN102735807B - Effect testing method for duck hemorrhagic ovaritis inactivated vaccine - Google Patents
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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, particularly relate to a kind of method of inspection of duck hemorrhagic oaritis inactivated vaccine effect, belong to veterinary biologics inspection technology field.
Background technology
Duck hemorrhagic oaritis (Duck Hemorrhagic Ovaritis, DHO) be a kind of Novel duck infectious disease of popular, acute, high degree in contact recently, its cause of disease is duck hemorrhagic oaritis virus (Duck Hemorrhagic Ovaritis Virus, DHOV), virus taxis belongs to the member of flaviviridae, infect different days duck, cause the ovarian hemorrhage of sexal maturity duck, orchiatrophy, egg production, fertility rate and hatchability to decline, duck death rate increases.
In the research of virus taxis status, by carrying out sequence homology analysis and evolutionary analysis to the F4 of isolated viral strain for the genome sequence of duck embryo passaged virus and known virus, find that the genetic distance of the tembusu virus of this virus and flaviviridae Flavivirus is nearest.In diagnosis, Cao Zhenzhen, Wan Chun and, Su Jingliang, Teng Qiao be vast etc., and people's report can adopt clinical symptoms, cardinal principle and histopathologic change, RT-PCR increases and the method such as virus purification makes a definite diagnosis this disease.About the prevention and control of this disease, 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 animal and veterinary research institute of academy of agricultural sciences of Chongqing City are separated during reporting and utilizing this disease to break out prepares the method for vaccine.Lin Jian, Liu Yuehuan etc. report the artificial challenge's model utilizing laying ducks successfully to establish duck hemorrhagic oaritis, and experiment basis has been established in the evaluation for vaccine.There is not yet the play-by-play of the vaccine potency evaluation method of objective, feasible, strong operability and science.
Summary of the invention
One of the object of the invention is to provide the age in days scope of a kind of duck hemorrhagic oaritis inactivated vaccine effect evaluation duck.
Two of the object of the invention is to provide the tissue samples of duck hemorrhagic oaritis inactivated vaccine effect evaluation vaccine immunity number of times, sampling time, collection.
Three of the object of the invention is to provide a kind of duck hemorrhagic oaritis inactivated vaccine effect evaluation method.
The present invention's virus stain called after used duck hemorrhagic oaritis virus HB strain, preserving number is CCTCC V201122; Described duck hemorrhagic oaritis virus is ssRNA virus, has cyst membrane, can aggegation dove red blood cell; Virion is spherical substantially, diameter 40 ~ 60nm; The resistibility of described virus environment is to external world comparatively strong, and in 4 DEG C, deposit a few week, in-20 DEG C, deposit some months, its appeal is all unaffected; Described virus is responsive to ether, chloroform; Most of detergent can by its rapid inactivation; Under 37 DEG C of conditions, fumigating 6h with 0.1% formalin just can its deactivation.This virus can 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 be cultivated on DEF and BHK21 cell.
The preparation method of the present invention's duck hemorrhagic used oaritis inactivated vaccine declared patent of invention in 2011, and application number is 201110255115.1.
The invention provides a kind of duck hemorrhagic oaritis inactivated vaccine method for testing efficacy, the method uses duck hemorrhagic oaritis inactivated vaccine immune duck, immunity 2 times altogether, within 2 weeks, carry out two after head exempts to exempt from, gather the two duck serum of exempting from latter 3 ~ 5 weeks and carry out ELISA antibody test, to be 0%, ELISA antibody positive rate be judged to vaccine potency be up to the standards for being not less than 80% nonimmune control group duck ELISA antibody positive rate.
Further, the inventive method, according to neurological susceptibility experimental result, determines that duck hemorrhagic oaritis inactivated vaccine efficacy test is 42 ~ 560 ages in days with the age in days of duck.
Further, in the inventive method 2 immune dosage be 0.5ml/ only ~ 2ml/ only.
Further, a kind of duck hemorrhagic oaritis inactivated vaccine method for testing efficacy provided by the invention uses duck hemorrhagic oaritis inactivated vaccine immunity 42 ~ 560 age in days duck, within 2 weeks, carry out two after head exempts to exempt from, gather the duck serum that two exempt from latter 3 ~ 5 weeks, use ELISA antibody assay kit to detect immune duck serum, carry out effect evaluation.
Further, the ELISA detection kit that above-mentioned ELISA antibody test is is envelope antigen with the flaviviridae of purifying.
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 uses duck hemorrhagic oaritis inactivated vaccine immunity 42 ~ 560 age in days duck, establishes the nonimmune control group duck of some simultaneously; Within 2 weeks, carry out two after head exempts to exempt from, the dosage of twice immunity is only 0.5ml/ ~ 2ml/, gather the duck serum that two exempt from latter 3 ~ 5 weeks, ELISA antibody assay kit is used to detect immune duck serum, carry out effect evaluation, nonimmune 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.
Further, the ELISA detection kit that above-mentioned ELISA antibody assay kit is is envelope antigen with the flaviviridae of purifying.
Further, described duck is that meat duck, egg are with duck or SPF duck.
The present invention proposes the tissue samples for duck hemorrhagic oaritis inactivated vaccine serology efficacy test uses the age in days of duck, vaccine potency checks the immune time of time vaccines, time of drawing materials, collection first, proposes the standard that serology ELISA vaccine potency test method, criterion and vaccine quality are qualified.Solve the blank of duck hemorrhagic oaritis inactivated vaccine serology effect evaluation method and criterion.The method that the present invention determines is objective, comprehensive, strong operability, can be used for laboratory and the clinical efficacy evaluation of duck hemorrhagic oaritis inactivated vaccine.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, biology used in embodiment, chemical reagent are conventional commercial, the conventional means that technological means used in embodiment is well known to those skilled in the art.Beijing duck used in embodiment is purchased from duck hemorrhagic oaritis negative antibody duck field (Beijing Golden Star Duck Center Nan Kou kind duck field), sheldrake is purchased from duck hemorrhagic oaritis negative antibody duck field (under the Pearl Spring of four seas town, Yanqing County of Beijing county dress circle village), SPF duck is hatched by the SPF duck's egg purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center, raises to the required age in days of test in SPF chicken isolator.
The test stone of susceptible duck
5 from without the immunity of duck hemorrhagic oaritis inactivated vaccine or have the duck of duck hemorrhagic oaritis popular history duck group, the inspection strain of every oral 1.0 milliliters of aseptic 0.5% lactoalbumin hydrolysate Hank ' s liquid 100 times dilutions of duck.Attack poison rear 4-5 day, blood sampling, separation of serum carries out virus purification.Virus isolated rate should be at least 4/5 positive, judges that inspection is susceptible duck with duck.
Embodiment 1 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) the efficacy test age in days of duck, immunizing dose, sampling time (1)
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201101, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 560 ages in days and do not infect the sheldrake 10 of DHOV, 0.5ml/ only, inject by chest muscle.Within 2 weeks, carry out two according to same dose and identical approach after head exempts to exempt from, the serum of 4 weeks ducks after collection secondary immunity, the ELISA antibody detection method of note is used to detect immune duck serum, nonimmune control group duck ELISA antibody positive rate is 0%, immune group duck ELISA antibody positive rate is 80%, illustrates that vaccine potency is up to the standards.
Embodiment 2 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) the efficacy test age in days of duck, immunizing dose, sampling time (2)
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 the Beijing duck 10 of DHOV, 1ml/ only, inject by chest muscle.Within 2 weeks, exempt from identical approach two according to same dose after head exempts from, the serum of 4 weeks ducks after collection secondary immunity, the ELISA antibody detection method of note is used to detect immune duck serum, nonimmune control group duck ELISA antibody positive rate is 0%, immune group duck ELISA antibody positive rate is 90%, illustrates that vaccine potency is up to the standards.
Embodiment 3 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) the efficacy test age in days of duck, immunizing dose, sampling time (3)
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201103, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 180 ages in days and do not infect the Beijing duck 10 of DHOV, 2ml/ only, inject by chest muscle.Within 2 weeks, exempt from identical approach two according to same dose after head exempts from, the serum of 4 weeks ducks after collection secondary immunity, the ELISA antibody detection method of note is used to detect immune duck serum, nonimmune control group duck ELISA antibody positive rate is 0%, immune group duck ELISA antibody positive rate is 100%, illustrates that vaccine potency is up to the standards.
Embodiment 4 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) the efficacy test age in days of duck, immunizing dose, sampling time (4)
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201104, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 560 ages in days and do not infect the SPF duck 10 of DHOV, 0.5ml/ only, inject by chest muscle.Within 2 weeks, carry out two according to same dose and identical approach after head exempts to exempt from, the serum of 3 weeks ducks after collection secondary immunity, the ELISA antibody detection method of note is used to detect immune duck serum, nonimmune control group duck ELISA antibody positive rate is 0%, immune group duck ELISA antibody positive rate is 80%, illustrates that vaccine potency is up to the standards.
Embodiment 5 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) the efficacy test age in days of duck, immunizing dose, sampling time (5)
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 the sheldrake 10 of DHOV, 1ml/ only, inject by chest muscle.Within 2 weeks, carry out two according to same dose and identical approach after head exempts to exempt from, the serum of 5 weeks ducks after collection secondary immunity, the ELISA antibody detection method of note is used to detect immune duck serum, nonimmune control group duck ELISA antibody positive rate is 0%, immune group duck ELISA antibody positive rate is 80%, illustrates that vaccine potency is up to the standards.
Embodiment 6 duck hemorrhagic oaritis inactivated vaccine (ELISA assay for antibodies) the efficacy test age in days of duck, immunizing dose, sampling time (6)
Use duck hemorrhagic oaritis inactivated vaccine (lot number 201106, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 400 ages in days and do not infect the Beijing duck 10 of DHOV, 2ml/ only, inject by chest muscle.Within 2 weeks, carry out two according to same dose and identical approach after head exempts to exempt from, the serum of 5 weeks ducks after collection secondary immunity, the ELISA antibody detection method of note is used to detect immune duck serum, nonimmune control group duck ELISA antibody positive rate is 0%, immune group duck ELISA antibody positive rate is 80%, illustrates that vaccine potency is up to the standards.
The preparation of embodiment 7 direct competitive-ELISA kit and TPPA
1. kit composition
2. material
2.1 biomaterial
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
Prepared by 2.3ELISA plate
2.3.1 the cell adapted Strain DHOV-HB strain of the purifying duck hemorrhagic oaritis (F5 generation) of virus inoculates DEF(DEF), when CPE occurs 85%, results virus.After freeze thawing 1 time, the centrifugal 15min of 3500rpm, gets supernatant, then through the centrifugal 30min of 10000rpm, get supernatant, concentrate through hollow fiber column, PEG6000 precipitates, sucrose density gradient centrifugation, gets the bright band between 30% and 45%, after the dilution of STE damping fluid and centrifugal treating, obtain purified virus.
2.3.2 virus bag is utilized the carbonate buffer solution (formula is shown in note 1) of pH9.6 by the dilution of 1000 times, virus, joins in 96 orifice plates, wrap by 12 ~ 16h under 4 DEG C of conditions with the dosage in 100ul/ hole.
2.3.3 wash plate washing lotion and wash plate 5 times, every hole is filled it up with.Every minor tick 30 seconds.
2.3.4 close and wash ELISA Plate after plate, pat dry.Add confining liquid 200ul.4 DEG C are spent the night, or 36 degree 1 hour.
2.3.5 dry closed rear ELISA Plate discards confining liquid, dries up rear for subsequent use.
2.3.6 Packaging Aluminum Foil encapsulation.
Horseradish peroxidase-labeled in duck hemorrhagic oaritis virus E protein monoclonal antibody, is prepared into the antibody that ultraviolet spectrophotometer 280nm place A value is about 2.5 enzyme conjugates marks by the preparation employing Over-voltage protection of 2.4 enzyme labelled antibodies.
The mensuration of 2.5 sample antibody to be checked
2.5.1 50 μ L sample diluting liquids are added at each Kong Zhongjun of elisa plate wrapping quilt;
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); 50 μ L test serums are added respectively in all the other each holes.
DEG C 2.5.321 ± 5 DEG C hatch 90min ± 6min.
2.5.4 remove each hole liquid, wash 3 times with 300 μ L washing fluids.
2.5.5 each hole adds the Horseradish Peroxidase Conjugates of 100 μ L 1 times dilution.
DEG C 2.5.621 ± 5 DEG C hatch 30min ± 3min.
2.5.7 remove each hole liquid, wash 3 times with 300 μ L washing fluids.
2.5.8 100 μ L substrate solutions are added in each hole.
2.5.9 lucifuge 21 DEG C ± 5 DEG C hatches 15min ± 2min.
2.5.10 in each hole, add 100 μ L stop buffer (2mol/L sulfuric acid) cessation reactions.
2.5.11 microplate reader is used to read under 450nm wavelength and record OD value.
Result judges
The OD value of negative control is greater than 0.700 test and sets up.Calculate the S/N value of each sample: S/N=sample OD value/negative serum OD value × 100, and according to the form below judges.
Points for attention
(1) substrate solution need keep in Dark Place, not with oxide interface.
(2) be preventing pollution, all in test used disposables need through autoclaving.
(3) critical value (cut-off) really regular inspection survey 300 parts of negative serums, calculate its fiducial limit of 95%.Be generally mean value X constant of negative control, this constant is generally will obtain according to 95% fiducial limit (normally the negative mean value of 2.1X).
The formula of kit solution of the present invention or reagent and preparation
(1) pH9.6 bag is buffered the formula of liquid (CB)
(2) formula of washing lotion
(3) formula of Block buffer
(4) formula of sample diluting liquid or enzyme mark dilution
(5) stop buffer 2mol/L sulfuric acid.
(6) formula of substrate solution A
The H of the formula 30% of substrate solution B
2o
2.
(7) 50 34 age in days SPF ducks are divided into 2 groups (infected group and non-infection control groups) by the preparation of positive and negative quality controlled serum at random, often organize 25.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.After superinfection 27 days, gather 2 groups of test duck blood, separation of serum, filtered, and often organizes serum mixing, adds thimerosal, quantitative separating, obtain 1 batch of positive serum and 1 batch of negative serum product in the ratio of total amount 0.01%.Positive serum and negative serum are all without bacteriological infection, and duck paramyxoviru, bird flu H5 hypotype, duck viral enteritis and duck hepatitis antibody are feminine gender, positive serum all can in and 100 ELD
50duck hemorrhagic oaritis virus, negative serum can not in and F4 generation 100 ELD
50duck hemorrhagic oaritis virus.
(8) preparation of DHOV-HB strain E protein monoclonal antibody is according to the primer of many strains flavivirus E gene that GenBank delivers, design pair of primers (F5-GCGAATTCATCCTCCTGCTGTTGGTC-3; R5-ATCTGGAGTGACGCCCATCCAGAGT-3), RT-PCR method amplification E gene order is adopted.The PCR primer reclaimed be connected with pMD18-T, transform DH5a competent cell, conventional method is extracted and is identified plasmid, by positive plasmid called after pET-E.PET-E is expressed in Amp+LB fluid nutrient medium, the E protein product that collection, qualification and purifying are expressed.By the E protein product Freund's complete adjuvant of purifying and incomplete Freund's adjuvant emulsification, preparation immunogene, immune BALB/c mouse, when E protein serum titer is qualified, gets spleen.SP2/0 myeloma cell and immune spleen cell are merged according to the ratio of 1:5, the positive hybridoma cell of screening and preparation secretion E protein antibody, clones positive hybridoma cell limiting dilution assay.By the positive colony hybridoma of above-mentioned some and paraffin oil injection mouse peritoneal, after 2 weeks, gather the ascites of mouse, be E protein monoclonal antibody.
Claims (2)
1. a duck hemorrhagic oaritis inactivated vaccine method for testing efficacy, is characterized in that, use duck hemorrhagic oaritis inactivated vaccine immunity 42 ~ 560 age in days duck, within 2 weeks, carry out two after head exempts from and exempt from, the dosage of twice immunity is only 0.5ml/ ~ 2ml/; Gather the duck serum that two exempt from latter 3 ~ 5 weeks, competitive ELISA antibody assay kit is used to detect immune duck serum, carry out effect evaluation, judge that whether the inspection of vaccine potency is qualified by nonimmune control group duck ELISA antibody positive rate and immune group duck ELISA antibody positive rate;
The ELISA detection kit that ELISA antibody assay kit used is is envelope antigen with the flaviviridae of purifying;
Enzyme labelled antibody used is duck hemorrhagic oaritis virus E protein monoclonal antibody;
Elisa plate envelope antigen used is the duck hemorrhagic oaritis virus HB strain totivirus of purifying, wraps quilt under 4 DEG C of conditions.
2. the method for claim 1, is characterized in that, described duck is that meat duck, egg are with duck or SPF duck.
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| CN103837682B (en) * | 2014-02-28 | 2016-08-10 | 北京市兽医实验诊断所 | A kind of duck novel banzi virus BYDV antibody ELISA detection kit |
| CN104215781A (en) * | 2014-09-04 | 2014-12-17 | 天津瑞普生物技术股份有限公司 | Porcine parvovirus (PPV) inactivated vaccine immune efficacy evaluation method |
| CN104977400B (en) * | 2015-07-27 | 2016-07-13 | 三明市和众生物技术有限公司 | A kind of sterility and infertility combined detection kit |
| CN105866424B (en) * | 2016-03-23 | 2017-05-24 | 北京市农林科学院 | Testing method for potency of vaccine against duck Tembusu viral diseases |
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| CN101773667A (en) * | 2010-01-28 | 2010-07-14 | 洛阳普莱柯生物工程有限公司 | Preparation method for vaccine of porcine circovirus II |
| CN102302772B (en) * | 2011-08-31 | 2015-11-18 | 齐鲁动物保健品有限公司 | A kind of duck hemorrhagic oophoritis inactivated vaccine and preparation method thereof |
| CN102488893B (en) * | 2011-12-28 | 2013-07-17 | 瑞普（保定）生物药业有限公司 | Duck hemorrhagic oophoritis inactivated vaccine production method by using cell line and product thereof |
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