CN102735853A - Method for determining titer of swine flu inactivated vaccine - Google Patents
Method for determining titer of swine flu inactivated vaccine Download PDFInfo
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- CN102735853A CN102735853A CN2012101861691A CN201210186169A CN102735853A CN 102735853 A CN102735853 A CN 102735853A CN 2012101861691 A CN2012101861691 A CN 2012101861691A CN 201210186169 A CN201210186169 A CN 201210186169A CN 102735853 A CN102735853 A CN 102735853A
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Abstract
The invention discloses a method for determining titer of a swine flu inactivated vaccine. The method comprises the following steps: immunizing a pig with a swine flue inactivated vaccine, collecting blood 21-28 days after immunization and separating blood serum; removing non-specific components in the serum to obtain a serum to be tested; diluting the swine flue inactivated antigen, and preparing a unit swine flue antigen diluent with a concentration of 4HA; and diluting the serum to be tested by multiple proportions, successively adding the 4HA unit swine flue antigen diluent and 1% red cells to conduct hemagglutination inhibition test, so as to completely inhibit highest dilution of the 4HA unit swine flu antigen serum at HI titer. The method has advantages of greatly shortened measurement time, little operation error, strong controllability, and small inter-batch difference, and also reduces detection errors caused by different levels of experimental animals in an animal challenge protection experiment for testing the titer.
Description
Technical field
The present invention
OnePlant the method for inspection of vaccine potency, particularly a kind of titration method of swine flu inactivated vaccine belongs to the veterinary biologics field.
Background technology
Swine flu (Swine Influenza, SI) be by the A of orthomyxovirus section type influenza virus cause a kind of acute, the height contagiousness the porcine respiratory infectious disease.Should disease be characteristic clinically with sudden cough, heating, expiratory dyspnea, depletion, rapid rehabilitation or death.This disease can betide the pig of each age and each kind, and the incidence of disease can be up to 100%.SI not only makes and suffers from the decline of pig production performance; Meat feed ratio reduces, and directly influences the swinery health status, causes that pig is only dead; And swine influenza virus has the close preferendum of height to the porcine respiratory epithelial cell; The respiratory tract natural cover for defense of pig body is destroyed, causes pig to breathe the concurrent or scabies secondary infections with various bacteria such as breeding syndrome virus, PRCV, pig pleuropneumonia actinomyces or virus, make epidemic situation more complicated and increase the weight of.In addition, SI still is one of main inhibitive ability of immunity disease of pig, can cause the huge waste of manual work, feed and medicine, causes enormous economic loss.
Using the inactivated vaccine inoculation is the effective way of control swine flu, however the present domestic commercial swine flu inactivated vaccine that still do not have, and the method for testing efficacy of confirming vaccine is the gordian technique that develops vaccine.Present domestic research unit is used to check the method for vaccine potency to attack malicious protection test, and this method required time is longer, and poor controllability, evaluation result are different because of the animal rank of using often, and assay also often has error.
Can't confirm tiring of swine flu vaccine quickly and accurately, become the difficult point of development swine flu vaccine.
Summary of the invention
The object of the present invention is to provide a kind of titration method of swine flu inactivated vaccine.
The technical scheme that the present invention taked is:
A kind of titration method of swine flu inactivated vaccine comprises the steps:
1) uses swine flu inactivated vaccine immune swine, the separation of serum of taking a blood sample after 21～28 days after the immunity;
2) nonspecific composition in the removal serum obtains serum to be checked;
3) with the dilution of swine flu inactivation antigen, compound concentration is 4 HA unit's swine flu antigenic dilutions;
4) with serum to be checked dilution, serum dilution being mixed with 4 HA unit's swine flu antigenic dilutions, red blood cell, carry out the blood clotting inhibition and test, is that HI tires with the high dilution of the serum that suppresses 4 HA unit's swine flu antigens fully.
Preferably, said method comprises the steps:
1) uses swine flu inactivated vaccine immune swine, the separation of serum of taking a blood sample after 21～28 days after the immunity;
2) serum is mixed with receptor destroying enzyme (RDE), nonspecific composition in the serum is removed in 37 ℃ of water-baths;
3) serum is moved to the residual RDE of 56 ℃ of water-bath 30min deactivation, add chicken red blood cell, 20～25 ℃ adsorb nonspecific composition remaining in the serum down;
4) it is centrifugal to have removed the serum of nonspecific composition, gets supernatant, and supernatant is serum to be checked;
5) the swine flu inactivation antigen being diluted, add chicken erythrocyte suspension afterwards, evenly under 20～25 ℃, leave standstill 20～30 min in the back in blood-coagulation-board concussion, is the blood clotting valency with the highly diluted multiple of the complete aggegation of red blood cell;
6) be 4 HA unit's swine flu antigenic dilutions according to the blood clotting valency compound concentration that records;
7) with serum to be checked dilution, adding 4 HA unit's swine flu antigenic dilution and chicken red blood cells, negative control is set simultaneously, carry out blood clotting and suppress experiment, is that HI tires with the high dilution of the serum that suppresses 4 HA unit's swine flu antigens fully.
Preferably, in the step 3), add chicken red blood cell and make that erythrocytic volumetric concentration is 5%～20% in the serum.
Preferably, step 5) and 7) in, the volumetric concentration of chicken red blood cell is 1%.
The invention has the beneficial effects as follows:
At first, the present invention has adopted the mensuration method that RCA suppresses to tire in the serum that RDE handles to come the immune efficacy of vaccine evaluation.This method has shortened minute greatly, and operate miss is little, and controllability is strong, and differences between batches are little, also reduced simultaneously with animal attack malicious protection test when coming testing effect usually because the animal used as test rank is different, the error that testing result often has.
Secondly, the assay method that RCA of the present invention suppresses to tire is easy, quick, have characteristics such as high specificity, can be used for the vaccine immunity efficacy test.
Embodiment
Below in conjunction with embodiment, further specify the present invention.
The acquisition of porcine blood serum and pre-service:
1) with 35 ages in days negative (5 of the susceptible piglets of HI subtype influenza HI antibody titer≤1:20), 2 injection swine flu inactivated vaccine (H1N1) 3 ml inoculated back 21 days, together with 5 blood samplings of contrast pig, separation of serum about each musculi colli divided;
2) for removing nonspecific composition in the serum, with serum and the volume ratio mixing of receptor destroying enzyme (RDE) by 1 ︰ 4, place 37 ℃ of water-bath effect 16～18h after, move to the residual RDE of 56 ℃ of water-bath 30min deactivation, take out the back and cool off;
3) add dense chicken red blood cell, making its final concentration is 5%～20%, at room temperature adsorbs 30min to remove other nonspecific composition;
4) low-speed centrifugal (2000r/min) 5～10min then draws supernatant, i.e. the test serum of 1 ︰, 5 dilutions is subsequent use.
Swine flu inactivated vaccine (H1N1) hemagglutination test (HA test):
1) gets 96 hole V-type Microhemagglutination plates; Every hole adds 25 μ l PBS, adds 25 μ l swine flu inactivation antigens in the 1st hole, lashes mixing repeatedly 8～10 times; Draw 25 μ l from the 1st hole and add the 2nd hole; Draw 25 μ l behind the mixing and add the 3rd hole, so carry out 2 times and be diluted to the 11st hole, the 11st hole is drawn 25 μ l and is discarded;
2) every hole adds 25 μ l1% chicken erythrocyte suspensions, and reaction plate is shaken 1～2min or gently detains the reaction plate mixed reactant on oscillator, under room temperature (20～25 ℃), leaves standstill 20～30 minutes;
3) result of determination when the control wells red blood cell significantly is button-type with reaction plate 60 degree that tilt, is observed red blood cell and is had or not the teardrop shape trickling, and the highly diluted multiple that does not have teardrop appearance trickling (100% aggegation) fully is the blood clotting valency.
The preparation of 4HA unit's antigen:
1), prepare 4HA unit's antigen with PBS according to the antigen blood clotting valency of measuring:
2) the 4HA unit's antigen that configures is diluted with PBS; Making its dilutability is 1:2,1:3,1:4,1:5,1:6,1:7, in each dilution 25 μ l antigen, adds 25 μ l PBS, adds 1% chicken erythrocyte suspension, 25 μ l again; Mixing, room temperature leave standstill 40min declares the result.
3) if the 1:4 dilution is 100% RCA terminal point, what show preparation is 4HA unit's antigen; If 100% RCA terminal point is 1:5 or 1:6, the 4HA unit's antigen that shows preparation is actually and is higher than 4 units; If 100% RCA terminal point is 1:2 or 1:3, the 4HA unit's antigen that shows preparation is actually and is lower than 4 units.Should suitably adjust according to assay, making the antigen working fluid is 4HA unit.
Hemagglutination-inhibition test (HI):
1) get 96 hole V-type micro-reaction plates, the 1st～11 hole adds 25 μ l PBS, and the 12nd hole adds 50 μ l PBS;
2) add 25 μ l serum to be checked in the 1st hole, fully draw 25 μ l behind the mixing in the 2nd hole, 2 times are diluted to the 10th hole successively, draw 25 μ l from the 10th hole and discard, and on every block of plate, establish standard positive serum and negative serum contrast;
3) the 1st～11 hole all adds the antigen of 25 μ l 4HA units, leaves standstill 30 min in room temperature;
4) add 25 μ l 1% (V/V) chicken erythrocyte suspensions in every hole, the concussion mixing leaves standstill 20～30 min in room temperature, and the contrast red blood cell will be button-type and be sunken at the bottom of the hole;
5) result judges: reaction plate is tilted, and the underflow person of dropping down is judged to the blood clotting inhibition to red blood cell from the hole at the same rate in all seroreactions hole and the red blood cell control wells, tires as HI with the high dilution of the serum that suppresses 4HA unit's antigen fully.
Vaccine is estimated
Tire as HI with the high dilution of the serum that suppresses 4HA unit's antigen fully.Be not less than 1:160 if back 21 days HI antibody mediated immunity groups of immunity have at least 4/5 pig HI only to tire, control group HI tires and should not be higher than 1:20, judges that then vaccine is qualified.
The checking of the inventive method reliability
Get 30 the 35 non-immune piglets of age in days and be divided into totally 3 groups of A, B, C at random, 10/group, criticize swine flu inactivated vaccine (H1N1) through musculi colli injection S0802 respectively; Wherein A organizes 0.3 ml/ head; B organizes 0.5 ml/ head, and C organizes 1 ml/ head, and other gets 5 with the not immune blank of doing of age in days piglet;
Back 21 days of immunity; Each group is gathered blood separation serum; Use receptor destroying enzyme (RDE) to handle serum in the ratio of 1:4; Measure HI with HI hypotype swine flu inactivation antigen and tire, and test pig is divided into totally four groups of I, II, III, IV, wherein the HI antibody titer≤1:40 of I group according to the height of HI antibody; HI antibody titer=the 1:80 of II group; HI antibody=the 1:160 of III group; HI antibody titer>=the 1:320 of IV group.
Attack malicious protection test
Above-mentioned I, II, III, IV totally four groups of immune swines are all attacked poison with the H1N1 hypotype swine influenza virus SGD01 strain that 1:10 doubly dilutes together with 5 with age in days negative antibody pig (control group), and each 1 ml/head of collunarium and intramuscular injection (contains 5 * 10 approximately
6.5EID
50), attack back 5 days each group collection nose swabs of poison and carry out the virus separation, use the nose swab that every pig gathered to put into respectively the 1ml sterile saline plastic centrifuge tube of (containing two resisting) is housed; After leaving standstill 3-4 hour under 4 ℃ of conditions; 3000rpm is centrifugal after allantoic cavity is inoculated 5 pieces of 9-11 age in days SPF chicken embryos, and every piece of 0.2ml is hatched for 35 ℃ and observed 72 hours; Measure chicken blastochyle blood clotting valency, be judged to infection with the blood clotting valency>=1:8 (micromethod) that has 1 piece of chicken embryo in 5 pieces of chicken embryos at least.With the negative chick embryo allantoic liquid blind passage of blood clotting valency once, decision method is the same.The result sees table 2.
Back 21 days HI antibody situation of immunity
H1N1 hypotype swine flu vaccine S0802 criticizes with 21 days HI antibody situation behind the non-immune piglet of various dose immunity 35 ages in days and sees table 1, and the result shows that the HI antibody titer mainly is distributed in 1:80 between the 1:160, accounts for 60%.
Experimental data shows, the method for inspection that swine flu inactivated vaccine of the present invention is renderd a service, and the result who obtains with the vaccine valence assay method of generally acknowledging has good consistance.
Attack malicious protection test
The result sees table 2, and test shows behind the vaccine immunity 21, uses the SGD01 strain to attack poison, and when HI antibody≤1:40, attacking malicious protection ratio is 0, and promptly vaccine can not be protected; When HI antibody=1:80, the malicious protection ratio of attacking of vaccine is 62.5%; And when HI antibody=1:160, vaccine attack malicious protection ratio more than 80%.Therefore we can think that HI antibody titer=1:160 is the protection lower limit of this vaccine, can be with the reference value of this numerical value as the vaccine potency check.Explained that also immune group has at least 4/5 pig HI only to tire and is not less than 1:160, control group HI tires and should not be higher than 1:20.Judge that then vaccine is qualified.
Claims (4)
1. the titration method of a swine flu inactivated vaccine comprises the steps:
1) uses swine flu inactivated vaccine immune swine, the separation of serum of taking a blood sample after 21～28 days after the immunity;
2) nonspecific composition in the removal serum obtains serum to be checked;
3) with the dilution of swine flu inactivation antigen, compound concentration is 4 HA unit's swine flu antigenic dilutions;
4) with serum to be checked dilution, serum dilution being mixed with 4 HA unit's swine flu antigenic dilutions, red blood cell, carry out the blood clotting inhibition and test, is that HI tires with the high dilution of the serum that suppresses 4 HA unit's swine flu antigens fully.
2. the titration method of swine flu inactivated vaccine according to claim 1 is characterized in that: said method comprises the steps:
1) uses swine flu inactivated vaccine immune swine, the separation of serum of taking a blood sample after 21～28 days after the immunity;
2) serum is mixed with receptor destroying enzyme (RDE), nonspecific composition in the serum is removed in 37 ℃ of water-baths;
3) serum is moved to the residual RDE of 56 ℃ of water-bath 30min deactivation, add chicken red blood cell, 20～25 ℃ adsorb nonspecific composition remaining in the serum down;
4) it is centrifugal to have removed the serum of nonspecific composition, gets supernatant, and supernatant is serum to be checked;
5) the swine flu inactivation antigen being diluted, add chicken erythrocyte suspension afterwards, evenly under 20～25 ℃, leave standstill 20～30 min in the back in blood-coagulation-board concussion, is the blood clotting valency with the highly diluted multiple of the complete aggegation of red blood cell;
6) be 4 HA unit's swine flu antigenic dilutions according to the blood clotting valency compound concentration that records;
7) with serum to be checked dilution, adding 4 HA unit's swine flu antigenic dilution and chicken red blood cells, negative control is set simultaneously, carry out blood clotting and suppress experiment, is that HI tires with the high dilution of the serum that suppresses 4 HA unit's swine flu antigens fully.
3. the titration method of swine flu inactivated vaccine according to claim 2 is characterized in that: in the step 3), add chicken red blood cell and make that erythrocytic volumetric concentration is 5%～20% in the serum.
4. the titration method of swine flu inactivated vaccine according to claim 2 is characterized in that: step 5) and 7), the volumetric concentration of chicken red blood cell is 1%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103336121A (en) * | 2013-06-13 | 2013-10-02 | 华中农业大学 | Swine influenza virus H3 subtype colloidal gold detection kit as well as preparation method and application thereof |
| CN107091935A (en) * | 2017-04-20 | 2017-08-25 | 陈凡 | Automate General layout Plan of blood clotting Inhibition test work station and application thereof |
| CN107328944A (en) * | 2017-06-14 | 2017-11-07 | 王琴 | A kind of blood clotting and hemagglutination-inhibition test screening technique |
| CN115656519A (en) * | 2022-10-25 | 2023-01-31 | 江苏乐汇生物技术有限公司 | Evaluation method capable of reducing nonspecific influence on serum titer of influenza vaccine |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1398325A1 (en) * | 2002-09-12 | 2004-03-17 | Wyeth | Monoclonal antibody specific for an epitope of inactivated feline immunodeficiency-encoded glycoprotein |
| CN101475641A (en) * | 2008-10-06 | 2009-07-08 | 浙江易邦生物技术有限公司 | Adenovirus vector avian influenza recombinant vaccine |
| CN101915837A (en) * | 2010-07-28 | 2010-12-15 | 中国兽医药品监察所 | Method for testing efficacy of hog cholera lapinized virus live vaccine |
| CN102399754A (en) * | 2011-05-27 | 2012-04-04 | 山东省农业科学院畜牧兽医研究所 | H9N2 avian influenza virus vaccine strain and application thereof in immune protection |
-
2012
- 2012-06-07 CN CN201210186169.1A patent/CN102735853B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1398325A1 (en) * | 2002-09-12 | 2004-03-17 | Wyeth | Monoclonal antibody specific for an epitope of inactivated feline immunodeficiency-encoded glycoprotein |
| CN101475641A (en) * | 2008-10-06 | 2009-07-08 | 浙江易邦生物技术有限公司 | Adenovirus vector avian influenza recombinant vaccine |
| CN101915837A (en) * | 2010-07-28 | 2010-12-15 | 中国兽医药品监察所 | Method for testing efficacy of hog cholera lapinized virus live vaccine |
| CN102399754A (en) * | 2011-05-27 | 2012-04-04 | 山东省农业科学院畜牧兽医研究所 | H9N2 avian influenza virus vaccine strain and application thereof in immune protection |
Non-Patent Citations (2)
| Title |
|---|
| 刘惠莉等: "猪流感病毒抗原表位基因表达载体构建与免疫原性分析", 《生物工程学报》, vol. 24, no. 4, 25 April 2008 (2008-04-25), pages 690 - 694 * |
| 王佑春: "疫苗效价检测方法的研究", 《中国生物制品学杂志》, vol. 20, no. 2, 20 February 2007 (2007-02-20), pages 150 - 152 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103336121A (en) * | 2013-06-13 | 2013-10-02 | 华中农业大学 | Swine influenza virus H3 subtype colloidal gold detection kit as well as preparation method and application thereof |
| CN103336121B (en) * | 2013-06-13 | 2015-03-04 | 华中农业大学 | Swine influenza virus H3 subtype colloidal gold detection kit as well as preparation method thereof |
| CN107091935A (en) * | 2017-04-20 | 2017-08-25 | 陈凡 | Automate General layout Plan of blood clotting Inhibition test work station and application thereof |
| CN107328944A (en) * | 2017-06-14 | 2017-11-07 | 王琴 | A kind of blood clotting and hemagglutination-inhibition test screening technique |
| CN115656519A (en) * | 2022-10-25 | 2023-01-31 | 江苏乐汇生物技术有限公司 | Evaluation method capable of reducing nonspecific influence on serum titer of influenza vaccine |
| CN115656519B (en) * | 2022-10-25 | 2024-05-10 | 江苏乐聚医药科技有限公司 | Evaluation method capable of reducing serum titer of influenza vaccine with nonspecific influence |
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