CN102735853B - 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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- CN102735853B CN102735853B CN201210186169.1A CN201210186169A CN102735853B CN 102735853 B CN102735853 B CN 102735853B CN 201210186169 A CN201210186169 A CN 201210186169A CN 102735853 B CN102735853 B CN 102735853B
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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
onethe method of inspection of planting vaccine potency, particularly a kind of titration method of swine flu inactivated vaccine, belongs to veterinary biologics field.
Background technology
Swine flu (Swine Influenza, SI) is the porcine respiratory infectious disease of caused by the A of orthomyxovirus section type influenza virus a kind of acute, height contagiousness.This disease is taking sudden cough, heating, expiratory dyspnea, exhaustion, rapid rehabilitation or death as feature clinically.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 affected pig production performance decline, meat feed ratio reduces, directly affect swinery health status, cause pig death, and swine influenza virus has the close preferendum of height to porcine respiratory epithelial cell, after infection, make the respiratory tract natural cover for defense of pig body destroyed, cause pig to breathe and the concurrent or scabies secondary infection of the various bacteria such as breeding syndrome virus, PRCV, pig pleuropneumonia actinomyces or virus, make epidemic situation more complicated with increase the weight of.In addition, one of inhibitive ability of immunity disease that SI or pig are main, can cause the huge waste of artificial, feed and medicine, causes huge economic loss.
Using inactivated vaccine inoculation is the effective way of controlling swine flu, but the current domestic commercial swine flu inactivated vaccine that there is no, the method for testing efficacy of determining vaccine is the gordian technique developing vaccine.Current domestic research unit is for checking the method for vaccine potency to have protest test, and the method required time is longer, poor controllability, and evaluation result is the animal rank difference because using often, and assay also often has error.
Cannot determine quickly and accurately tiring of swine flu vaccine, 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 solution used in the present invention is:
A titration method for swine flu inactivated vaccine, comprises the steps:
1) use swine flu inactivated vaccine immune swine, the separation of serum of taking a blood sample after 21～28 days after immunity;
2) remove the nonspecific composition in serum, obtain serum to be checked;
3) by the dilution of swine flu inactivation antigen, compound concentration is 4 HA unit's swine flu antigenic dilutions;
4) by serum to be checked dilution, serum dilution is mixed with 4 HA unit's swine flu antigenic dilutions, red blood cell, carry out blood clotting inhibition and test, tire taking the high dilution of serum that suppresses 4 HA unit's swine flu antigens completely as HI.
Preferably, described method comprises the steps:
1) use swine flu inactivated vaccine immune swine, the separation of serum of taking a blood sample after 21～28 days after immunity;
2) serum is mixed with receptor destroying enzyme (RDE), nonspecific composition in serum is removed in 37 DEG C of water-baths;
3) serum is moved to the residual RDE of 56 DEG C of water-bath 30min deactivation, add chicken red blood cell, adsorb nonspecific composition remaining in serum at 20～25 DEG C;
4) by centrifugal the serum of having removed nonspecific composition, get supernatant, supernatant is serum to be checked;
5) swine flu inactivation antigen is diluted, add afterwards chicken erythrocyte suspension, after blood-coagulation-board concussion evenly, at 20～25 DEG C, leave standstill 20～30 min, taking the highly diluted multiple of the complete aggegation of red blood cell as blood clotting valency;
6) be 4 HA unit's swine flu antigenic dilutions according to the blood clotting valency compound concentration recording;
7) by serum to be checked dilution, add 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, tire taking the high dilution of serum that suppresses 4 HA unit's swine flu antigens completely as HI.
Preferably, in step 3), adding chicken red blood cell to make erythrocytic volumetric concentration in serum is 5%～20%.
Preferably, step 5) and 7) in, the volumetric concentration of chicken red blood cell is 1%.
The invention has the beneficial effects as follows:
First, the present invention has adopted the mensuration method that red cell agglutination suppresses to tire in the serum of RDE processing to evaluate the immune efficacy of vaccine.The method has shortened minute greatly, and operate miss is little, and controllability is strong, and differences between batches are little, has also reduced while carrying out testing effect with animal protest test usually because of animal used as test rank difference the error that testing result often has simultaneously.
Secondly, red cell agglutination of the present invention suppress the assay method of tiring easy, quick, there is the features such as high specificity, can be used for vaccine immunity efficacy test.
Embodiment
Below in conjunction with embodiment, further illustrate the present invention.
the acquisition of pig serum and pre-service:
1), with 5 of 35 age in days feminine genders (HI subtype influenza HI antibody titer≤1:20) susceptible piglets, each musculi colli divides 2 of left and right injection swine flu inactivated vaccine (H1N1) 3 ml, inoculates latter 21 days, together with 5 blood samplings of contrast pig, separation of serum;
2) for removing nonspecific composition in serum, serum and receptor destroying enzyme (RDE) are mixed by the volume ratio of 1 ︰ 4, be placed in after 37 DEG C of water-bath effect 16～18h, move to the residual RDE of 56 DEG C of water-bath 30min deactivation, cooling after taking out;
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, and the test serum that 1 ︰ 5 dilutes is for subsequent use.
swine flu inactivated vaccine (H1N1) hemagglutination test (HA test):
1) get 96 hole V-type Microhemagglutination plates, every hole adds 25 μ l PBS, add 25 μ l swine flu inactivation antigens in the 1st hole, repeatedly lashing 8～10 times mixes, draw 25 μ l from the 1st hole and add the 2nd hole, mix rear absorption 25 μ l 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, reaction plate is shaken on oscillator to 1～2min or gently detains reaction plate mixed reactant, under room temperature (20～25 DEG C), leaves standstill 20～30 minutes;
3) result of determination in the time that control wells red blood cell is significantly button-type, by reaction plate 60 degree that tilt, observes red blood cell and has or not teardrop shape trickling, is blood clotting valency completely without the highly diluted multiple of teardrop sample trickling (100% aggegation).
the preparation of 4HA unit's antigen:
1) according to the antigen blood clotting valency of measuring, with PBS preparation 4HA unit antigen:
2) the 4HA unit's antigen PBS configuring is diluted, 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, then adds 1% chicken erythrocyte suspension 25 μ l, mix, room temperature leaves standstill 40min and sentences result.
3) if 1:4 dilution is 100% red cell agglutination terminal, what show preparation is 4HA unit's antigen; If 100% red cell agglutination terminal is 1:5 or 1:6, show that 4HA unit's antigen of preparation is actually higher than 4 units; If 100% red cell agglutination terminal is 1:2 or 1:3, show that 4HA unit's antigen of preparation is actually lower than 4 units.Should suitably adjust according to assay, making antigen working fluid is 4HA unit.
hemagglutination-inhibition test (HI):
1) get 96 hole V-type micro-reaction plates, 1st～11 holes add 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 mix rear absorption 25 μ l in the 2nd hole, 2 times are diluted to the 10th hole successively, draw 25 μ l and discard, and on every block of plate, establish standard positive serum and negative serum contrast from the 10th hole;
3) 1st～11 holes all add the antigen of 25 μ l 4HA units, leave standstill 30 min in room temperature;
4) in every hole, add 25 μ l 1%(V/V) chicken erythrocyte suspension, concussion mixes, and leaves standstill 20～30 min in room temperature, at the bottom of contrast red blood cell will be button-type and be sunken to hole;
5) result is judged: reaction plate is tilted, and in all seroreactions hole and red blood cell control wells, red blood cell is judged to blood clotting inhibition from the hole underflow person of dropping down at the same rate, tires as HI using the high dilution of serum that suppresses 4HA unit's antigen completely.
vaccine is evaluated
Tire as HI using the high dilution of serum that suppresses 4HA unit's antigen completely.Be not less than 1:160 if latter 21 days HI antibody mediated immunity groups of immunity have at least the HI of 4/5 pig to tire, control group HI tires should be higher than 1:20, judges that vaccine is qualified.
the checking of the inventive method reliability
Get 30 nonimmune piglets of 35 age in days and be divided at random totally 3 groups of A, B, C, 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, separately gets 5 with the not immune blank of doing of age in days piglet;
Latter 21 days of immunity, each group gathers blood separation of serum, use receptor destroying enzyme (RDE) to process serum in the ratio of 1:4, measuring HI with HI hypotype swine flu inactivation antigen tires, and according to the height of HI antibody, test pig is divided into totally four groups of I, II, III, IV, wherein the HI antibody titer≤1:40 of I group; 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.
protest test
Above-mentioned I, II, III, IV totally four groups of immune swines are attacked poison together with 5 H1N1 hypotype swine influenza virus SGD01 strains of all doubly diluting with 1:10 with age in days negative antibody pig (control group), and the each 1 ml/head of collunarium and intramuscular injection is (approximately containing 5 × 10
6.5eID
50), attack latter 5 days each group collection nose swabs of poison and carry out virus separation, use respectively the nose swab that every pig gathered to put into the 1ml sterile saline plastic centrifuge tube of (containing dual anti-) is housed, under 4 DEG C of conditions, leave standstill after 3-4 hour, after 3000rpm is centrifugal, through 5 pieces of allantoic cavity inoculation 9-11 age in days SPF chicken embryos, every piece of 0.2ml, hatches for 35 DEG C and observes 72 hours, measure chicken blastochyle blood clotting valency, to have blood clotting valency>=1:8(micromethod of 1 piece of chicken embryo in 5 pieces of chicken embryos at least) be judged to infection.With the negative chick embryo allantoic liquid blind passage of blood clotting valency once, decision method is the same.The results are shown in Table 2.
latter 21 days HI antibody situations of immunity
H1N1 hypotype swine flu vaccine S0802 criticizes with the HI antibody situation of 21 days after the nonimmune piglet of various dose immunity 35 age in days in table 1, and result shows that HI antibody titer is mainly distributed in 1:80 between 1:160, accounts for 60%.
Experimental data shows, the method for inspection of swine flu inactivated vaccine effect of the present invention, and the result obtaining with generally acknowledged vaccine valence assay method has good consistance.
protest test
The results are shown in Table 2, test shows after vaccine immunity 21, uses SGD01 strain to attack poison, and in the time of HI antibody≤1:40, attacking malicious protection ratio is 0, and vaccine can not be protected; In the time of HI antibody=1:80, the malicious protection ratio of attacking of vaccine is 62.5%; And in the time of 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, the reference value that can check this numerical value as vaccine potency.Also illustrated that immune group has at least the HI of 4/5 pig to tire and is not less than 1:160, control group HI tires should be higher than 1:20.Judge that vaccine is qualified.
Claims (4)
1. a titration method for swine flu inactivated vaccine, comprises the steps:
1) use swine flu inactivated vaccine immune swine, blood sampling separation of serum on the 21st～28 after immunity;
2) remove the nonspecific composition in serum, obtain serum to be checked;
3) by the dilution of swine flu inactivation antigen, compound concentration is 4 HA unit's swine flu antigenic dilutions;
4) by serum to be checked dilution, serum dilution is mixed with 4 HA unit's swine flu antigenic dilutions, red blood cell, carry out blood clotting inhibition and test, tire taking the high dilution of serum that suppresses 4 HA unit's swine flu antigens completely as HI.
2. the titration method of swine flu inactivated vaccine according to claim 1, is characterized in that: described method comprises the steps:
1) use swine flu inactivated vaccine immune swine, blood sampling separation of serum on the 21st～28 after immunity;
2) serum is mixed with receptor destroying enzyme (RDE), nonspecific composition in serum is removed in 37 DEG C of water-baths;
3) serum is moved to the residual RDE of 56 DEG C of water-bath 30min deactivation, add chicken red blood cell, adsorb nonspecific composition remaining in serum at 20～25 DEG C;
4) by centrifugal the serum of having removed nonspecific composition, get supernatant, supernatant is serum to be checked;
5) swine flu inactivation antigen is diluted, add afterwards chicken erythrocyte suspension, after blood-coagulation-board concussion evenly, at 20～25 DEG C, leave standstill 20～30 min, taking the highly diluted multiple of the complete aggegation of red blood cell as blood clotting valency;
6) be 4 HA unit's swine flu antigenic dilutions according to the blood clotting valency compound concentration recording;
7) by serum to be checked dilution, add 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, tire taking the high dilution of serum that suppresses 4 HA unit's swine flu antigens completely as HI.
3. the titration method of swine flu inactivated vaccine according to claim 2, is characterized in that: in step 3), adding chicken red blood cell to make erythrocytic volumetric concentration in serum is 5%～20%.
4. the titration method of swine flu inactivated vaccine according to claim 2, is characterized in that: step 5) and 7) in, the volumetric concentration of chicken red blood cell is 1%.
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| 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 |
| CN107328944B (en) * | 2017-06-14 | 2019-02-12 | 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 |
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