CN104215781A - Porcine parvovirus (PPV) inactivated vaccine immune efficacy evaluation method - Google Patents
Porcine parvovirus (PPV) inactivated vaccine immune efficacy evaluation method Download PDFInfo
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Abstract
The invention provides a porcine parvovirus (PPV) inactivated vaccine immune efficacy evaluation method. According to the method, the immune efficacy of a porcine parvovirus inactivated vaccine is evaluated by inspecting a serum antibody level of an organism after being injected with the vaccine. The scheme is characterized in that the evaluation method for the efficacy of an ELISA antibody of PPV is innovatively established. The method has characteristics of simplicity in operation, specificity, sensitivity and the like, and an effective technical measure is provided for detecting the PPV inactivated vaccine antibody.
Description
Technical field
The present invention relates to technical field of vaccines, be specifically related to a kind of porcine parvovirus inactivated vaccines immune efficacy evaluation method.
Background technology
Pig parvoviral (Porcine parvovirus, PPV) causes the just Reproduction Disorder of pregnant sow, and main manifestations is infection and the death of embryo and tire pig, and sow is usually expressed as inferior clinical symptom.Serology feminine gender just pregnant sow is less than 70 days infection PPV in the gestational period, can cause the infection of embryo or tire pig, thereby produce the breeding difficulty symptoms such as miscarriage, stillborn foetus, mummy tire.PPV is at ubiquity all over the world, become at present and caused embryo and tire pig to infect and one of dead main pathogen.This virus is except directly causing sow breeding difficulty, also relevant with piglet dermatitis to the inflammatory diarrhea of piglet.In addition, PPV and porcine circovirus 2 type mixed infection, bringing out postweaning multisystemic wasting syndrome also has report.
The evaluation method of the porcine parvovirus inactivated vaccines effect using at present, is classical hemagglutination-inhibition test.Experimental animal used is pig or cavy, and because PPV negative antibody pig is difficult to look for, and experimentation cost is high again, selects the effect evaluation that replaces pig to complete vaccine to the cavy of PPV sensitivity to obtain expert's accreditation.For the use of cavy, before test, must measure in cavy body whether have PPV HI antibody, only have PPV HI negative antibody cavy, the effect that could be used for PPV inactivated vaccine detects, after cavy contact PPV, in body, will produce PPV antibody, therefore, before each test, all should carry out corresponding monitoring screening, ensure to use PPV negative antibody cavy, in each test, also to set up negative control group, in case infect PPV in process of the test.In these processes, we will and measure antibody by HI experiment sieving, blood serum sample need to carry out the processing of white bole etc., to the judgement of blood clotting valency, and the demarcation of 4 HA of unit, test PBS liquid PH control etc., whole process of the test is relatively high to operator's requirement, and the cycle of test is longer, and the human factor impact of result is larger, the method mostly can only be served as qualitative or relative quantification, cannot carry out accurate quantification.Reagent and GPRBC that this process is used affect the evaluation to vaccine effect.
Summary of the invention
The present invention is intended to the technological deficiency for prior art, and a kind of porcine parvovirus inactivated vaccines immune efficacy evaluation method is provided, and short to realize determination period, result easily judges, the technical purpose that cost is low.
For realizing above technical purpose, the present invention by the following technical solutions:
A kind of porcine parvovirus inactivated vaccines immune efficacy evaluation method, comprises the following steps:
1) preparation standard negative serum and standard positive serum:
Gather the guinea pig serum of PPV HI < 1:8, after white bole solution-treated as standard female serum; Serum after the negative cavy of selection inactivated vaccine immunity, through white bole solution-treated, blood clotting suppresses the serum of valency > 1:64, as standard positive serum;
2) concentrated, the purifying of viral antigen;
3) antigen is coated;
4) detect:
In ELISA Plate, add the test serum 100 μ l after dilution, 37 DEG C of effect 0.5～1.5h;
Add coated PPV purifying antigen, 37 DEG C of effect 0.5～1.5h;
Discard primary antibodie, wash with PBST;
Add the HRP mark sheep anti-mouse igg 100 μ l of PBS dilution, 37 DEG C of effect 0.5～1.5h;
Discard two anti-ly, wash with PBST;
Add nitrite ion to carry out lucifuge colour developing 15～25min;
Every hole adds 50 μ l stop buffer color development stopping;
Microplate reader reads OD
450value.
Preferably, described step 2) comprise the following steps:
Utilize IBRS-2 or ST cell culture and virus, reach 80% when above until cytopathy, results virus-culturing fluid, multigelation results save backup; Virus-culturing fluid, through 4 DEG C of centrifugal 30min of 5000rmp/min, is gone to precipitation, and supernatant through 4 DEG C of centrifugal 60min of 10000rpm/min, is got supernatant again; Supernatant packs in bag filter uses 0.01M pH7.4PBS damping fluid, and dialysis 3-4d, changes liquid every day 4 times; Then concentrated with PEG6000, obtain viral concentrate, save backup in-20 DEG C.
Preferably, step 3) comprise the following steps:
With positive serum and the OD of negative serum under different antigen concentrations
450the maximum the suitableeest serum dilution of standard as selected the suitableeest serum dilution of the difference of value, with antigen diluent degree corresponding to this dilutability for the suitableeest antigen coated concentration; To antigen, execution is coated with for condition to utilize the suitableeest above-mentioned serum dilution and the suitableeest antigen coated concentration.
Preferably, step 3) to carry out antigen coated concentration when antigen coated be 15～30ug/mL.
Preferably, step 3) to carry out the extension rate of positive serum when antigen coated be 1:4～1:16.
In above technical scheme as testing result, i.e. OD
450value>=2.1 o'clock are differentiated for antibody positive, vaccine has produced effect; As testing result, i.e. OD
450when value < 2.1, differentiate is that negative antibody, vaccine do not produce effect.
The present invention has following technological merit:
(1) detection method sense cycle of the present invention is short, detect that result judgement only needed about 3 hours, and HI inhibition test needs 4～6 hours detection time at present.
(2) the method stability is better than HI method, simple to operate, favorable repeatability, and differences between batches are stable.
(3) specificity is good, can accurately judge yin and yang attribute result, and < 2 can only be detected when at present HI tires screening mouse
3below, the ELISA method of setting up by the present invention can be determined negative mouse accurately, the negative mouse of screening that can be stricter.
(4) evaluation of antagonist level, ELISA method testing result and HI titration result coincidence rate that the present invention sets up reach 100%.
The present invention adopts the parvovirus antigen of purifying, with the coated elisa plate of virus, then react with the cavy positive serum of guinea pig serum antibody to be checked and preparation, then with anti-reaction of the anti-cavy two of pig of horseradish peroxidase-labeled, finally develop the color by TMB, utilize the rear blood sampling of GB product porcine parvovirus inactivated vaccines immune guinea pig (PPV HI<8) separation of serum of buying simultaneously, prepare the positive criteria serum that is directed to PPV, gather negative cavy (PPV HI<8) serum as negative control simultaneously, utilize microplate reader testing result.For the screening of the negative cavy of potency test at parvovirus inactivated vaccine, and the evaluation of antibody horizontal.The method and conventional blood clotting suppress (HI) test relatively: determination period is short, and result more easily judges, susceptibility is higher, and specificity is better.The ELISA that this research is set up has the features such as easy and simple to handle, special and responsive, for the antibody test of PPV inactivated vaccine cavy potency test provides a kind of effective technology means.
Embodiment
The screening of cavy for embodiment 1 porcine parvovirus inactivated vaccines evaluation
1. buying of cavy: buy 10 cavys, derive from Beijing Vital River Experimental Animals Technology Co., Ltd..
2. blood sampling separation of serum, carries out respectively the mensuration of ELISA antibody and HI antibody titer.
2.1HI antibody titer is measured
2.1.1 antigen preparation: GPRBC blood clotting valency is reached to the above and viral level of 1:128 and reach 10
5tCID
50when/0.1ml, through formalin-inactivated, after sterilizing thoroughly as hemagglutination-inhibition test antigen.With 50% red cell agglutination (HA
50) the high dilution of antigen judge terminal as antigen valence result.Antigen HA
50tire and be not less than 1:128.
2.1.2 the manufacture of positive serum and negative serum control sample: select healthy susceptible piglet of 3-6 monthly age, gathering that determination of serum HI tires is that 0 serum contrasts as negative serum.Select to prepare serum after the immune pig of inactivated vaccine (BJ-2 strain), an one antigen workload (is 4HA
50) carrying out hemagglutination-inhibition test mensuration, tire>=1:128 of HI, as positive control serum.
2.1.3 pig parvoviral blood clotting and anti-pig parvoviral serum antibody hemagglutination-inhibition test operation steps
2.1.3.1 hemagglutinin working fluid preparation
1. hemagglutinin determination of agglutination titer: hemagglutinin is diluted to different multiples with physiological saline, adds 0.5% GPRBC suspension.96 orifice plates are shaken up on oscillator, be placed in 20-30 DEG C, 20-40min result of determination, makes the high dilution of 50% red cell agglutination, as judging terminal.
Table 1 hemagglutinin determination of agglutination titer flow data
2. the configuration of hemagglutinin working fluid and inspection
4HA
50the configuration of hemagglutinin: if hemagglutinin 50% determination of agglutination titer result is 1:1024,4 50% HAUs (being 4HA50)=1024/4 (being 1:256).Get physiological saline 9ml, add hemagglutinin 1ml, 1:10 dilution.Get this dilution 1ml and join in 24.6ml, making ultimate density is 1:256.
Inspection: the 1:256 of preparation times dilution is added to physiological saline 1ml with the amount of 1ml respectively, 2ml, 3ml, 4ml, 5ml, in 6ml, making its whole dilutability is 1:2,1:3,1:4,1:5,1:6,1:7.Then use 96 hole micro plates, add 0.5% GPRBC suspension 0.025ml from each dilution liquid 0.025ml, then add physiological saline 0.025ml, mix.Blood-coagulation-board is placed after 20-40min in room temperature, is 4HA50 if configure annoying antigen liquid, and 1:4 dilutability will provide 50% aggegation terminal; If 4HA50 is higher than 4 units, possible 1:5 or 1:6 are terminal; If lower, possible 1:2 or 1:3 are terminal.Should adjust.
2.1.4 hemagglutination-inhibition test
1. the preparation of 25% white bole suspension: get white bole 25g, put in centrifuge tube, add appropriate 15mol/L hydrochloric acid solution, fully shake up, with the centrifugal supernatant of abandoning of 2000rmp10min, then add the hydrochloric acid solution of 1mol/L, shake up, then centrifugal treating.3 times so repeatedly, the white bole PBS that finally gets precipitation is mixed with 25% suspension, adjust after pH value is 7.2 with 5mol/LNaOH solution, 15 pounds of 20min autoclavings processing (121 DEG C of 10min), deposit in 4 DEG C for subsequent use, storage life is no more than 4 months.
2. 0.5% GPRBC: use without 3 times (3000rmp10min) of calcium magnesium PBS washing, then to become 0.5% red cell suspension without calcium magnesium PBS solution preparation.(50ul+9950ul is without the PBS solution of calcium ions and magnesium ions).
3. the processing of serum to be checked: 0.1ml serum+0.6ml25% to be checked white bole suspension+0.1ml Chinese krebs solution mixes, put room temperature 20min, shake 2-3 time therebetween, 2000rmp10min is centrifugal, in supernatant, adds 20%-50% GPRBC suspension 50ul, shake up, put room temperature 20min, for several times, 2000rmp10min is centrifugal in shake therebetween, get supernatant, be serum to be checked for hemagglutination-inhibition test (having diluted 1:8).
4. HI method: every row adds Chinese krebs solution 0.025ml/ hole (the first hole does not add)+serum 0.025ml/ to be checked hole (9-12 hole does not add), 2 times of doubling dilution to the 8 holes.Every 1-11 hole of arranging adds the 4 hemagglutinin 0.025ml/ of unit holes, and the 12nd hole does not add hemagglutinin and adds 0.05ml Chinese krebs solution dilution.The 9th hole is that the contrast of pig parvoviral negative serum, the 10th hole are the contrast of pig parvoviral positive serum, and the 11st hole is hemagglutinin contrast, and the 12nd hole is red blood cell contrast.Vibration mixes 10-15 second, puts 37 DEG C of 1h, adds 0.05ml GPRBC/hole, and vibration mixes, and puts 37 DEG C of effect 20-40min or under room temperature, after 2h, observes result of determination.
Table 2 hemagglutination-inhibition test data
Result of determination: using last 1 hole without obviously agglutinating reaction (suppressing completely) as terminal.
The mensuration of 2.2ELISA antibody titer
2.2.1 control serum sample preparation: PPV positive serum is prepared by the immune HI negative antibody of PPV inactivated vaccine (BJ-2 strain) (purchased from Ruipu (Baoding) Biological Pharmaceutical Co., Ltd.) (or ELISA negative antibody) cavy, HI up to standard being decided to be: the 1:128 that tires, as vaccine, evaluation is used with reference to positive serum, the negative guinea pig blood that gathers PPV HI<8, separation of serum is as negative control sera.
2.2.2 main solution: 1. coating buffer CBS (0.05M carbonate buffer solution, pH9.6); 2. cleansing solution (PBST, pH7.4); 3. confining liquid: containing 5% skimmed milk; 4. substrate solution (TMB); 5. stop buffer: 2moL/L H
2sO
4solution.
2.2.3 indirect ELISA operation steps
1. every hole adds serum to be checked 100 μ l after treatment, comprises positive and negative control wells (repeating 2 holes), 37 DEG C of effect 1h;
2. add in the ELISA ELISA Plate that is coated with PPV purifying antigen;
3. 37 DEG C of effect 1h;
4. discard primary antibodie, PBST washing;
5. HRP mark sheep anti-mouse igg (two is anti-) the 100 μ l that add PBS dilution, put into 37 DEG C of incubators, effect 1h;
6. discard two anti-ly, wash with PBST;
7. add nitrite ion to carry out lucifuge colour developing 20min;
8. every hole adds 50 μ l stop buffer color development stopping;
9. microplate reader reads OD
450;
10. result decision method: serum OD to be checked
450/ with the OD of dilutability negative control sera
450>=2.1 are judged to positive findings; Serum OD to be checked
450/ with the OD of dilutability negative control sera
450< 2.1 is judged to negative findings.
3. interpretation of result: the ELISA method measurement result that the present invention sets up and HI inhibition test measurement result are coincide, and by serum OD to be checked
450/ with the OD of dilutability negative control sera
450value can determine yin and yang attribute cavy completely,, along with the interference that is determined with human factor of carrying out test findings of test, there is false-negative result in and HI inhibition test, as shown in table 3.
The ELISA of table 3 guinea pig serum measures and HI measurement result
Embodiment 2: porcine parvovirus inactivated vaccines effect evaluation is measured
1. the screening of cavy and immunity: 10 of the cavys that is 350-400g by body weight (PPV HI antibody titer: < 1:8 or ELISA negative antibody), be divided into 3 groups, 8 of immunity, intramuscular injection vaccine 0.5ml respectively, after 28d, together with 2 of contrast cavys, blood sampling separation of serum, measures respectively PPV HI antibody and ELISA antibody.Requiring contrast guinea pig serum negative, should all there is antibody response in immune guinea pig, and serum HI tires and answers >=1:64.
2HI antibody titer is measured (referring to embodiment 1,2.1HI antibody titer assay method carries out).
The mensuration (referring to embodiment 1,2.2ELISA antibody titer assay method carries out) of 3.ELISA antibody titer.
4. interpretation of result: ELISA measures with HI titration and obtains consistent results, positive coincidence rate 100%.
Show that the method can finely be applied in the effect evaluation of vaccine.
After table 4 vaccine immunity, the ELISA of guinea pig serum measures and HI measurement result
The specificity of embodiment 3:ELISA antibody titer assay method and replica test research
One, test method
(1) specific test
1. blocking test: PPV positive serum is fully mixed with the PPV virus liquid of purifying, 37 DEG C of effect 1.5h, the centrifugal 20min of 5000r/min, gets supernatant and carries out ELISA detection.Change according to the OD value of sample before and after blocking-up the specificity of determining test.
2. intercrossing test: with the PPV antigen wrapper sheet of purifying, for detection of 6 kinds of other viruses (CSFV, porcine reproductive and respiratory syndrome virus, PRV, Latex agglutination test, pig circular ring virus) positive serum of preparing inactivated vaccine immune guinea pig adds in the ELISA Plate of envelope antigen and carries out ELISA test, establish feminine gender and blank, observations simultaneously.
(2) replica test: choose at random 20 parts of serum after PPV immune guinea pig, carry out ELISA respectively at the different time and test duplicate detection, observations.
(3) sensitivity tests: randomly draw 10 parts of PPV immune serums, detect the relatively result of two kinds of tests according to the indirect ELISA method of setting up and PPV HI test simultaneously.
Two, test findings
(1) specific test
Blocking test: detect respectively the P/N value before and after positive serum blocking-up prepared by cavy parvovirus vaccine by the indirect EL ISA method of setting up, find that the positive serum ELISA testing result after blocking-up is all negative, illustrate that PPV Antigen Using can block positive serum and react with envelope antigen, show that the ELISA setting up is specific.
Intercrossing test: after the positive serums such as CSFV, PRRSV, PRV, JEV, PCV-2 are processed, detect by the indirect ELISA method of setting up, and establish PPV positive serum and negative serum contrast.Other cause of disease serum of result OD value is all suitable with PPV negative serum OD value, and P/N value is all less than 2.1, and positive serum OD
450value is 0.285, P/N value > 2.1.The above results shows, cross reaction does not occur for PPV antigen and other cause of disease serum.
Table 5PPV antigen and different virus antigen are prepared the cross matching result (OD of serum
450)
(2) revision test: cavy blood examination and 20 parts of porcine parvovirus inactivated vaccines immune serums respectively at the different time to 40 parts of purchases carry out ELISA detection, found that fluctuating is no more than 0.05 for testing result (P/N value) with a serum, prove that the indirect ELISA method of setting up has good repeatability.
(3) sensitization test: 10 parts of cavy immune serums and 10 parts of not immune guinea pig serum are carried out respectively to PPV HI test and ELISA detection, result is as shown in table 6, have consistent result by the visible ELISA test of table with HI test, and the testing result of two kinds of methods has good correlativity.
The sensitivity tests result of table 6 negative serum
The sensitivity tests result of table 7 positive serum
In sum, the ELISA method that the present invention sets up is no matter in to the shaker test of negative cavy, or in vaccine potency evaluation test, all show with HI and test identical testing result, coincidence rate reaches 100%, in sense cycle, and the about 3h of the whole process of ELISA method left and right, and HI method, at preparation 4HA
50after antigen, often, due to the difference that 50% agglutination titer is judged, cause 4HA
50again the preparation of antigen, whole process completes needs 4-6 hour, and the foundation of the method has greatly reduced the test period, also reduces because testing crew reduces the error causing.
Confirm by above embodiment, the ELISA method that the present invention sets up has good specificity, repeatability.
In the application that the present invention evaluates at vaccine potency, can accurately judge by the size of ELISA value the gradient of infection of negative cavy to the screening of negative cavy, and HI tests because serum will pass through kaolinic processing, result of determination can only reach < 2
3below, this must make to understand because the use of some false negative cavys affects vaccine evaluation effect in follow-up vaccine evaluation.
Can the testing result of indirect ELISA reflect the infection state of cavy and the immune effect of vaccine exactly, depends primarily on its specificity, and wherein the purity of envelope antigen is again the specific principal element of impact.The antigen coated ELISA Plate of this PPV that tests to purify, by the screening of optimum reaction condition, and the checking of blocking test, cross matching, revision test, tentatively set up the antibody indirect ELISA method that detects PPV inactivated vaccine immune guinea pig.Being based upon of the method can be used as to a certain extent the method that substitutes HI and completes vaccine potency evaluation, in the screening of the negative cavy of test and immune efficacy are evaluated, all has good application prospect.
Above embodiments of the invention are had been described in detail, but described content is only preferred embodiment of the present invention, not in order to limit the present invention.All any amendments of making in application range of the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (5)
1. a porcine parvovirus inactivated vaccines immune efficacy evaluation method, is characterized in that comprising the following steps:
1) preparation standard negative serum and standard positive serum:
Gather the guinea pig serum of PPV HI < 1:8, after white bole solution-treated as standard female serum; Serum after the negative cavy of selection inactivated vaccine immunity, through white bole solution-treated, blood clotting suppresses the serum of valency > 1:64, as standard positive serum;
2) concentrated, the purifying of viral antigen;
3) antigen is coated;
4) detect:
In ELISA Plate, add the test serum 100 μ l after dilution, 37 DEG C of effect 0.5～1.5h;
Add coated PPV purifying antigen, 37 DEG C of effect 0.5～1.5h;
Discard primary antibodie, wash with PBST;
Add the HRP mark sheep anti-mouse igg 100 μ l of PBS dilution, 37 DEG C of effect 0.5～1.5h;
Discard two anti-ly, wash with PBST;
Add nitrite ion to carry out lucifuge colour developing 15～25min;
Every hole adds 50 μ l stop buffer color development stopping;
Microplate reader reads OD
450value.
2. a kind of porcine parvovirus inactivated vaccines immune efficacy evaluation method according to claim 1, is characterized in that step 2) comprise the following steps:
Utilize IBRS-2 or ST cell culture and virus, reach 80% when above until cytopathy, results virus-culturing fluid, multigelation results save backup; Virus-culturing fluid, through 4 DEG C of centrifugal 30min of 5000rmp/min, is gone to precipitation, and supernatant through 4 DEG C of centrifugal 60min of 10000rpm/min, is got supernatant again; Supernatant packs in bag filter uses 0.01M pH7.4PBS damping fluid, and dialysis 3-4d, changes liquid every day 4 times; Then concentrated with PEG6000, obtain viral concentrate, save backup in-20 DEG C.
3. a kind of porcine parvovirus inactivated vaccines immune efficacy evaluation method according to claim 1, is characterized in that step 3) comprise the following steps:
With positive serum and the OD of negative serum under different antigen concentrations
450the maximum the suitableeest serum dilution of standard as selected the suitableeest serum dilution of the difference of value, with antigen diluent degree corresponding to this dilutability for the suitableeest antigen coated concentration; To antigen, execution is coated with for condition to utilize the suitableeest above-mentioned serum dilution and the suitableeest antigen coated concentration.
4. a kind of porcine parvovirus inactivated vaccines immune efficacy evaluation method according to claim 1, is characterized in that step 3) to carry out antigen coated concentration when antigen coated be 15～30ug/mL.
5. a kind of porcine parvovirus inactivated vaccines immune efficacy evaluation method according to claim 1, is characterized in that step 3) to carry out the extension rate of positive serum when antigen coated be 1:4～1:16.
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| CN111551747A (en) * | 2020-05-15 | 2020-08-18 | 金宇保灵生物药品有限公司 | Method for testing efficacy of porcine epikavirus inactivated vaccine by using rabbit based on antibody detection |
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| 李斐等: "猪细小病毒VP2蛋白主要抗原域间接ELISA方法的建立", 《中国生物工程杂志》 * |
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| CN105004856A (en) * | 2015-07-10 | 2015-10-28 | 青岛易邦生物工程有限公司 | Porcine circovirus II antibody detection ELISA kit |
| CN107589256A (en) * | 2017-09-30 | 2018-01-16 | 天津瑞普生物技术股份有限公司 | The method of inspection of the type of Haemophilus parasuis 4,5 type bivalent inactivated vaccine effect |
| CN108220254A (en) * | 2018-01-30 | 2018-06-29 | 扬州大学镇江高新技术研究院 | A kind of porcine reproductive and respiratory syndrome virus purification process |
| CN108918870A (en) * | 2018-07-07 | 2018-11-30 | 广东省实验动物监测所 | The liquid-phase chip detection method of pig parvoviral |
| CN111551747A (en) * | 2020-05-15 | 2020-08-18 | 金宇保灵生物药品有限公司 | Method for testing efficacy of porcine epikavirus inactivated vaccine by using rabbit based on antibody detection |
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