CN103336116A - Method for detecting efficacy of classical swine fever live vaccines - Google Patents
Method for detecting efficacy of classical swine fever live vaccines Download PDFInfo
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Abstract
The invention belongs to the technical field of novel biological medicines of veterinary medicines, and in particular relates to a method for detecting the efficacy of classical swine fever live vaccines. According to the method, the efficacy of live vaccines can be evaluated by detecting the virus content of live vaccines by an indirect immunofluorescence (IFA) method. The method is short in detection time, simple to operate, low in cost, accurate in result and good in repeatability, and has a good application value and wide market prospect.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of live vaccines of hog cholera method for testing efficacy.
Background technology
Swine fever (Classical Swine fever, abbreviation CSF) is called hog cholera (Hog cholera) or Europe class swine fever (European swine fever) again, it is the viral infectious disease of the acute or super febris acuta of a boar, feature clinically is to disseminate rapidly, have a fever, and can see typical hemorrhage pathology when dissecting check.The pig that swine fever can infect the various ages only, popular throughout the year, M ﹠ M is all high, and is very harmful to pig.This disease is to threaten one of most important infectious disease of pig industry.OIE (OIE) classifies swine fever one of as 16 kinds of Notifiable diseases of category-A, and China then is decided to be a class deadly infectious disease.Many countries and regions use live vaccines of hog cholera to force immunoprophylaxis, also are the best approaches of preventing and treating swine fever at present.
At present, China's production live vaccines of hog cholera effectiveness detection method all is to adopt rabbit precursor reactant by the use of thermal means.Not only need much to test White Rabbit, time-consuming effort again, and because minimizing gradually and the strain of China's pure lines large ear rabbit are more and more assorted, cause nowadays being difficult to accurately detect with rabbit precursor reactant heat the effectiveness of live vaccine.
Summary of the invention
Given this, the invention provides a kind of new live vaccines of hog cholera method for testing efficacy, specifically, by detecting the effectiveness that live vaccine viral level in passage cell is estimated live vaccine with indirect immunofluorescence (IFA) method, substitute traditional rabbit precursor reactant by the use of thermal means, with reach quick and precisely, the simple and effective swine fever purpose of vaccine potency while still alive that detects.
The invention provides a kind of live vaccines of hog cholera method for testing efficacy, comprise the steps:
(1) prepares passage cell
To cover with the cell of individual layer, disperse with the EDTA-trypsinization, obtain the passage cell suspension;
(2) viral level of live vaccines of hog cholera in passage cell measured
Return the freeze-drying live vaccines of hog cholera molten and dilution with cell maintenance medium, cell suspension in live vaccine dilution and the step (1) is added in the Tissue Culture Plate, place incubator to cultivate, set up simultaneously not connect malicious normal cell contrast;
(3) indirect immunofluorescence (IFA) method detects viral level in step (2) the gained cell
1. Tissue Culture Plate is taken out from incubator, discard and cultivate liquid in the plate hole, wash with PBS;
2. adding aqueous acetone solution fixes;
3. discard aqueous acetone solution, wash with PBS;
4. the swine fever positive serum (primary antibodie) that adds the PBS dilution;
5. discard primary antibodie, the PBS washing;
6. the anti-pig IgG of FITC mark rabbit (two is anti-) that adds the PBS dilution;
7. discard two and resist, with the PBS washing, add PBS at last and preserve;
8. place and observe the fluorescence situation under the fluorescent microscope, judge live vaccines of hog cholera effectiveness.
Preferable, in cell plates, add in the cell suspension in the step (1), add diluted live vaccines of hog cholera liquid in the step (2).
Preferable, the dilution of live vaccines of hog cholera described in the step (2) is to carry out under 0-8 ℃ of low temperature environment.
Preferable, incubator is CO in the step (2)
2Incubator, temperature are 36-37 ℃, CO
2Content is 2.5%-5%, and incubation time is 2-5 days.
Preferable, the number of times of PBS washing is 2-7 time in the step (3).
Preferable, the aqueous acetone solution temperature is 0-8 ℃ in the step (3), concentration is 30%-90%.
Preferable, the extension rate range of choice of primary antibodie is 50-1000 times in the step (3), two anti-extension rate ranges of choice are 50-10000 times.
Preferably, the extension rate of primary antibodie is 400 times in the step (3), and two anti-extension rates are 500 times.
Compared with prior art, the present invention has following technique effect:
1. lack detection time, only need 3 days, need 11 days at least and detect with rabbit body infective dose usually.
2. use cell infection virus, simple to operate, cost is low, does not need to buy a large amount of animals used as test, does not need special messenger's determination experiment animal heat simultaneously.
3. utilize cell infection virus, the experimental result significant difference that causes the animal used as test individual difference can not appear and in condition homogeneous, stable.
Embodiment
Embodiment 1
The live vaccines of hog cholera method for testing efficacy
1. preparation passage cell
Pig testis (ST) cell that covers with individual layer (is derived from ATCC, U.S. typical case bacterium kind preservation center), pancreatin with 0.25% (containing 0.02% EDTA) digestion disperses the back to add the α-MEM cell maintenance culture solution that contains 3% hyclone, after the cell count, cell suspension is joined in the 96 porocyte plates every hole 100 μ l.
2. the dilution of live vaccines of hog cholera
Present embodiment has prepared three mass products vaccines, and has measured its antigenic content respectively.
The strain of live vaccines of hog cholera is hog cholera lapinised virus strain (China Veterinery Drug Inspection Office's preservation, preserving number AV1412).With hog cholera lapinised virus strain inoculation ST cell, the viral antigen liquid of gathering in the crops is equipped with suitable freeze drying protectant makes live vaccines of hog cholera.With the α that contains 3% hyclone-MEM cell maintenance culture solution with freeze-drying live vaccines of hog cholera Hui Rong, on ice or other can keep carrying out 10 times of serial dilutions in 0-8 ℃ the low temperature environment, get 10
-1-10
-7Dilutability adds in 96 orifice plates of the Tissue Culture Plate in the step 1, and every hole 100 μ l, each dilutability do 8 holes and repeat, and Tissue Culture Plate is placed CO
2Cultivate in the incubator, temperature is 36-37 ℃, CO
2Content is 2.5%-5%, and incubation time is 2-5 days.Preferably, temperature is 37 ℃, CO
2Content is 5%, incubation time is 3 days, can carry out indirect immunofluorescence (IFA) and judge malicious valency.Simultaneously, set up and do not connect the contrast of malicious normal cell.Concrete steps are as follows:
(1) discarding liquid in 96 orifice plates, is 7.4 with the PBS(pH value of 0.01mol/L) wash 2-7 time, 3min at every turn, optimally, washing times is 3 times, at every turn 3min;
(2) adding temperature is 0-8 ℃ 30%-90% aqueous acetone solution, optimally, 80% aqueous acetone solution 100 μ l/ holes, 4 ℃ of refrigerators are 30min fixedly;
(3) discarding acetone, is 7.4 with the PBS(pH value of 0.01mol/L) wash 2-7 time, each 3min, optimally, washing times is 3 times, each 3min;
(4) the PBS(pH value that adds with 0.01mol/L is 7.4) make the swine fever positive serum (primary antibodie is available from China Veterinery Drug Inspection Office) that 50-1000 doubly dilutes, optimally, extension rate is 400 times, antibody content 5 μ g/ml, every hole 100 μ l, 37 ℃ of effect 60min;
(5) discarding primary antibodie, is 7.4 with the PBS(pH value of 0.01mol/L) wash 2-7 time, each 3min, optimally, washing times is 3 times, each 3min;
(6) the PBS(pH value that adds with 0.01mol/L is 7.4) make the anti-pig IgG of fluorescein isothiocynate (FITC) mark rabbit (two is anti-, available from Sigma company) that 50-10000 doubly dilutes, optimally, extension rate is 500 times, antibody content 6 μ g/ml, every hole 100 μ l, 37 ℃ of effect 60min;
(7) discarding two anti-ly, is 7.4 with the PBS(pH value of 0.01mol/L) wash 3 times, 10min at every turn, the PBS(pH value that adds 0.01mol/L at last is 7.4), every hole 50 μ l;
(8) place observation fluorescence situation under the fluorescent microscope, record fluorescence hole count is calculated TCID according to the Reed-Muench method
50The result;
(9) IFA fluorescence criterion: special yellow-green fluorescence does not appear in not connecing in the malicious normal control cell cytosol of setting up, and can be observed specific yellow-green fluorescence and connect in the malicious cell plate hole inner cell endochylema, is judged to be positive hole.Observations such as following table 1.
Table 1 indirect immunofluorescence (IFA) observations
Calculating TCID50 result according to the Reed-Muench method the following is shown in the table 2.
The malicious valency measurement result of table 2 three mass products vaccines
| Vaccine batch | (every part contains TCID to poison valency measurement result 50） |
| I is criticized | 10 4.5TCID 50/ head part |
| II is criticized | 10 4.667TCID 50/ head part |
| III is criticized | 10 4.57TCID 50/ head part |
3. animal is attacked the poison experiment
(1) live vaccine dilution: concrete dilution process following (every bottle of vaccine calculates by 20 parts):
I, II, III are criticized vaccine, two bottles of freeze dried vaccines are with physiological saline mixing Hui Rong to 40ml, every part 1ml, get the vaccine liquid that 1ml wherein returns after molten and add 5.325ml, 8.290ml, 6.431ml physiological saline respectively, and then carry out 10 times of serial dilutions and 5 times of dilutions successively, the most every batch of vaccine dilution is viral level 5TCID
50/ ml, 1TCID
50/ ml, 0.2TCID
50The vaccine of/ml uses liquid.Concrete operations are as follows:
Get dilution back vaccine liquid 1ml and add 9ml physiological saline; Get step dilution back vaccine liquid 1ml and add 9ml physiological saline; Get step dilution back vaccine liquid 1ml and add 9ml physiological saline; Get step dilution back vaccine liquid 2ml and add 8ml physiological saline; Get step dilution back vaccine liquid 2ml and add 8ml physiological saline.
(2) animal experiment design
This test is divided into 10 groups at random with 39 piglets (swine fever antigen antibody is even, the health of negative, body weight all), and attacking malicious control group is 3,4 every group of immune group.Every batch of vaccine is distinguished immune 3 groups, and immunizing dose is respectively 0.2TCID
50/ head part, 1TCID
50/ head part, 5TCID
50/ head part.Back 14 days of immunity, immune group injects 10 together with the identical control group of condition
5The swine fever crossdrift of minimum lethal dose (MLD) is blood poison 1ml (10
5MLD refers to that with the swine fever crossdrift be blood poison dilution 10
5Doubly malicious 1ml is attacked to pig in the back, and pig is still dead, is the malicious standard of attacking in the industry).Observed 16, result of determination, as shown in table 3 below.According to the result, add up malicious valency and attack correlativity between the poison protection, count minimum immune dosage.
The design of table 3 animal experiment and result
Attack malicious result of experiment and show, I, II, III are criticized live vaccines of hog cholera (ST passage cell) and are diluted to 1TCID respectively
50/ ml, injection 1ml can reach the total protective number of piglet.Namely 3 mass products vaccines are 1TCID to the minimum immune dosage of the strong poison of swine fever crossdrift system
50/ head part.At present the hog cholera vaccine national standard all is still can resist strong virus attack with immune swine after 3000 times of the vaccine dilutions an of part, and according to the inventive method, when the live vaccines of hog cholera that detects is tired more than or equal to 10
3.5TCID
50During/head part, dilute 3000 times after immune swine, can reach total protection effect, meet national examination criteria.Therefore, process according to the invention can propose to identify that live vaccines of hog cholera meets the standard that country is detected.
In sum, the invention provides a kind of new live vaccines of hog cholera method for testing efficacy, specifically, by detecting the effectiveness that live vaccine viral level in passage cell is estimated live vaccine with indirect immunofluorescence (IFA) method.This method is short detection time, and simple to operate, cost is low, the result that obtains is accurate, favorable repeatability has good using value and vast market prospect.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (7)
1. a live vaccines of hog cholera method for testing efficacy is characterized in that: comprise the steps:
(1) prepares passage cell
To cover with the cell of individual layer, disperse with the EDTA-trypsinization, obtain the passage cell suspension;
(2) viral level of live vaccines of hog cholera in passage cell measured
Return the freeze-drying live vaccines of hog cholera molten and dilution with cell maintenance medium, cell suspension in live vaccine dilution and the step (1) is added in the Tissue Culture Plate, place incubator to cultivate, set up simultaneously not connect malicious normal cell contrast;
(3) indirect immunofluorescence (IFA) method detects viral level in step (2) the gained cell
1. Tissue Culture Plate is taken out from incubator, discard and cultivate liquid in the plate hole, wash with PBS;
2. adding aqueous acetone solution fixes;
3. discard aqueous acetone solution, wash with PBS;
4. the swine fever positive serum (primary antibodie) that adds the PBS dilution;
5. discard primary antibodie, the PBS washing;
6. the anti-pig IgG of FITC mark rabbit (two is anti-) that adds the PBS dilution;
7. discard two and resist, with the PBS washing, add PBS at last and preserve;
8. place and observe the fluorescence situation under the fluorescent microscope, judge live vaccines of hog cholera effectiveness.
2. live vaccines of hog cholera method for testing efficacy according to claim 1 is characterized in that: add in cell plates in the cell suspension in the step (1), add diluted hog cholera vaccine liquid in the step (2).
3. live vaccines of hog cholera method for testing efficacy according to claim 1 is characterized in that: the dilution of live vaccines of hog cholera described in the step (2) is carried out under 0-8 ℃ of low temperature environment.
4. live vaccines of hog cholera method for testing efficacy according to claim 1 is characterized in that: incubator is CO in the step (2)
2Incubator, temperature are 36-37 ℃, CO
2Content is 2.5%-5%, and incubation time is 2-5 days.
5. live vaccines of hog cholera method for testing efficacy according to claim 1 is characterized in that: the number of times of PBS washing is 2-7 time in the step (3).
6. live vaccines of hog cholera method for testing efficacy according to claim 1 is characterized in that: the aqueous acetone solution temperature is 0-8 ℃ in the step (3), and concentration is 30%-90%.
7. live vaccines of hog cholera method for testing efficacy according to claim 1 is characterized in that: in the step (3) the extension rate range of choice of primary antibodie be 50-1000 doubly, two anti-extension rate ranges of choice be 50-10000 doubly.
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| CN201310239545.3A CN103336116B (en) | 2010-06-22 | 2010-06-22 | A kind of method for testing efficacy of swine fever live vaccine |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103698518A (en) * | 2013-12-30 | 2014-04-02 | 山东滨州博莱威生物技术有限公司 | Method for detecting virus content of swine fever live vaccine through indirect immunofluorescence |
| CN104407136A (en) * | 2014-12-07 | 2015-03-11 | 青岛易邦生物工程有限公司 | Method for measuring content of viruses in avian encephalomyelitis live vaccine |
| CN104535764A (en) * | 2014-12-07 | 2015-04-22 | 青岛易邦生物工程有限公司 | Classical swine fever virus gene recombinant adenovirus vector vaccine virus content determination method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102600469A (en) * | 2012-03-07 | 2012-07-25 | 齐鲁动物保健品有限公司 | Classical swine fever live vaccine |
| CN103908665B (en) * | 2013-01-05 | 2016-05-25 | 普莱柯生物工程股份有限公司 | A kind of vaccine combination and its preparation method and application |
| CN103105493A (en) * | 2013-02-07 | 2013-05-15 | 新疆天康畜牧生物技术股份有限公司 | Novel method for replacing detection of rabbit thermal response of hog cholera lapinized virus strain |
| CN103616505B (en) * | 2013-12-10 | 2016-01-27 | 瑞普（保定）生物药业有限公司 | A kind of hog cholera lapinised virus live vaccine effect detection method |
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| CN104407136A (en) * | 2014-12-07 | 2015-03-11 | 青岛易邦生物工程有限公司 | Method for measuring content of viruses in avian encephalomyelitis live vaccine |
| CN104535764A (en) * | 2014-12-07 | 2015-04-22 | 青岛易邦生物工程有限公司 | Classical swine fever virus gene recombinant adenovirus vector vaccine virus content determination method |
Also Published As
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|---|---|
| CN101846683A (en) | 2010-09-29 |
| CN101846683B (en) | 2014-08-06 |
| CN103336116B (en) | 2016-01-13 |
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