CN102004151B - Avian influenza virus H9 subtype hemagglutination inhibition antigen standard substance and preparation method thereof - Google Patents
Avian influenza virus H9 subtype hemagglutination inhibition antigen standard substance and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an avian influenza virus H9 subtype hemagglutination inhibition antigen standard substance and a preparation method thereof. The preparation method comprises the following steps: preparing a virus liquid of avian influenza virus H9 CKSH01 strain virus, and inspecting; inactivating the virus liquid, concentrating, and inspecting the semi-finished product; and freeze-drying, inspecting the finished product, inspecting the uniformity and stability, calibrating the standard substance, fixing the value and the like to obtain the standard substance. The standard substance is the basic guarantee for accurate diagnosis and immunity monitoring of avian influenza virus H9 and the proper evaluation on the vaccine immunity effect, and enhances the level for preventing and controlling avian influenza.
Description
Technical field
The present invention relates to a kind of avian influenza virus H9 hypotype blood clotting and suppress antigen standard substance and preparation method, belong to veterinary biologics field.
Background technology
The chemical reference substance of British Pharmacopoeia (BP) proposed ability from 1963, because WHO establishes international biological standard material center in Britain, so Britain continues to use international reference materials always, and its domestic standard material is set up more late, after 1970, European Pharmacopoeia comes out, and Britain is except quoting international reference materials, also European Pharmacopoeia standard material is used, a small amount of standard substance using this country.Even so, BP1968 version and enlarged edition thereof add quantity that British Pharmacopoeia (nineteen sixty-eight) records again more than 260, and within 1980, version is more than 300, and BP1993 version has recorded 371.The American Pharmacopeia council can provide about more than 1805 kind of pharmaceutical standard material; European Pharmacopoeia Commission has 2098 kinds, and wherein biological product standards material has 319 kinds.
At present, China has developed national standard reference material 1262 kinds, and secondary reference material 1739 kinds, relates to the fields such as iron and steel, geology, oil, nuclear material, environment, food and clinical examination.The standard substance research of veterinary biologics is more delayed, only have 192 kinds at present, and veterinary biologics inspection standard substance is almost nil.
Whether accurately veterinary biologics standard substance controls and determine veterinary biologics product quality, calibration verification testing instruments and method standard of physical, is the material base correctly diagnosing infectious disease, accurate measurements Immunity.
Bird flu (AI) is the great animal epidemic of China's emphasis prevention and control, and avian influenza virus (AIV) H9 hypotype is popular very extensive.In current bird flu diagnosis, immunologic surveillance and vaccine immunity effect evaluation thereof, most widely used method is HI test, this is the method that the Ministry of Agriculture of China in 2006 approves, but China there is no the report of avian influenza virus H9 hypotype hemagglutination-inhibition test standard substance and correlative study thereof at present, the reference laboratory such as external International Animal Health tissue (OIE) do not have yet, and before and after having had a strong impact on, different times detects the consistance of data and the comparability of different experiments number of chambers certificate.In recent years laboratory at different levels gets more and more to the demand of bird flu standard substance, the diagnostic reagent standard substance that particularly H9 hypotype is relevant with H5 hypotype, therefore developing avian influenza virus H9 hypotype blood clotting suppresses antigen standard substance particularly urgent, this standard substance is bird flu H9 hypotype Accurate Diagnosis, immunologic surveillance and carry out the correct basic guarantee evaluated to its immune effect of vaccine, improves the prevention and control level of bird flu.
Development Primary Reference and " standard substance management method " and the primary standard material technical manual (JJG 1006-94) of following State Metrological Bureau's issue enforcement on July 10th, 1987 of current China standard substance, but this technical manual is the development being applicable to chemical composition, physicochemical characteristics and engineering characteristic primary standard material, for biological product standards material, particularly veterinary biologics Developments of certified reference samples there is no technical manual at present and can follow.Therefore veterinary biologics Developments of certified reference samples is a footless research, has filled up the blank in the research field.
Summary of the invention
The present invention is a kind of avian influenza virus H9 hypotype blood clotting suppression of preparation antigen standard substance, and this antigen standard substance contains the avian influenza virus H9 subtype C KSH01 strain virus of deactivation; Hemagglutination-inhibition test antigen answers >=7log2 to chicken red blood cell agglutination titer; This antigen can only suppress by avian influenza virus H9 hypotype positive serum, and to H_5 subtype, H7 hypotype positive serum, newcastle disease positive serum and Egg Drop syndrome virus positive serum are all negative reaction.
Main preparation methods of the present invention is:
(1) prepare avian influenza virus H9 hypotype hemagglutination-inhibition test antigen standard substance material standed for, comprising: the preparation of H9 subtype avian influenza virus seed culture of viruses and inspection; The preparation of virus liquid and inspection; Virus liquid deactivation, concentrated, the inspection of semifinished product; Freeze-drying, product inspection, Homogeneity Test and stability test; (2) demarcation of standard substance, definite value.
Elaborating of technical solution of the present invention
Comprise avian influenza virus H9 hypotype hemagglutination-inhibition test antigen standard substance, its preparation method and program, it prepares the content such as code and cooperation proving operation specification.
The present inventor is on existing avian influenza virus H9 hypotype (strain) the blood clotting suppressing method basis of country, quality standard and preparation code that avian influenza virus H9 hypotype blood clotting suppresses antigen standard substance are formulated, have developed avian influenza virus H9 hypotype blood clotting and suppress antigen standard substance, and tire to have carried out cooperating to it with 8 units such as China Veterinary Drugs Supervisory Inst., China Agricultural University and demarcate.
One, avian influenza virus H9 hypotype (strain) blood clotting suppresses the preparation of antigen standard substance
1 viral seed culture of viruses
(1) source of viral seed culture of viruses
The present invention suppresses the virus stain of antigen standard substance to be A type avian influenza virus A/Chicken/Shanghai/10/01 (H9N2) strain (being called for short CKSH01 strain) for the manufacture of avian influenza virus H9 hypotype (strain) blood clotting, is identified, takes care of and supply by China national bird flu reference laboratory (in Harbin Veterinary Medicine Inst., China Academy of Agriculture of Chinese Harbin City).
(2) characteristic of viral seed culture of viruses
1) red cell agglutination valency carries out chicken red blood cell determination of agglutination titer by annex 1, and seed culture of viruses answers >=8log2 to 1% chicken red blood cell agglutination titer.
2) seed culture of viruses sterile saline is made 10 times of serial dilutions by viral level, gets 10
-6, 10
-7, 10
-83 dilutabilitys, inoculation 10 age in days SPF chicken embryo 5 in each allantoic cavity, every embryo 0.1ml.Put 37 DEG C to continue to hatch, chicken embryo dead before 24h discards to be disregarded, and chicken embryo dead in 24 ~ 72h takes out and at any time in 2 ~ 8 DEG C of stored refrigerated.To 72h, measure the red cell agglutination valency of all chicken blastochyles, agglutination titer>=4log2 person is judged to infection, calculates EID
50(the Chinese veterinary pharmacopoeia council. People's Republic of China's veterinary drug allusion quotation in 2005 version three. Chinese agriculture publishing house, 2006, the present invention is hereinafter referred to as " Chinese veterinary pharmacopoeia "), every ml viral level should>=10
7.5eID
50.
3) seed culture of viruses sterile saline is diluted to 10 by specificity
3eID
50/ 0.1ml mixes with equivalent avian influenza virus H9 subtype sepcific positive serum, puts in room temperature and after 1h, inoculates 10 age in days SPF chicken embryo 10, observing 120h.Should not cause specific death in 24 ~ 120h after inoculation, have at least 8/10 inoculated into chick embryo strong alive, chicken blastochyle does hemagglutination test (HA test), should be negative.
4) by seed culture of viruses sterile saline, 10 times of dilutions are done to the virulence of chicken, collunarium inoculation SPF chicken in 4 ~ 5 week age 10, every 0.1ml.The SPF chicken 5 that condition of separately getting is identical, does not inoculate in contrast, raises respectively under same condition, observes 14, and infected chicken and contrast chicken all should not occur any abnormal response.
5) method that pure property specifies by " Chinese veterinary pharmacopoeia " should be polluted without bacterium, mould, mycoplasma and exogenous virus.
6) within basic bacteria algebraically 5 generation.
7) seed culture of viruses is kept at less than-70 DEG C, and storage life is 60 months.
2 animals used as test
The SPF chicken used and SPF chicken embryo provide by Harbin veterinary institute Experimental Animal Center.
3 avian influenza virus H9 hypotype (strain) blood clottings suppress the preparation of antigen standard substance material standed for
(1) preparation of antigen
Conventionally (" Chinese veterinary pharmacopoeia "), seed culture of viruses is inoculated SPF chicken embryo, results chicken blastochyle is prepared avian influenza virus H9 hypotype (strain) blood clotting and is suppressed antigen semi-manufacture, through deactivation, concentrates, after carrying out steriling test, titration, by every bottle of 1ml packing freeze-drying.The formula that protective agent adopts this problem to develop and compound method (preparing sucrose concentration with water for injection is the freeze drying protectant of 100%, and after filtration sterilization, protective agent and chicken blastochyle are by 1: 9 proportions), lot number is 200901.
(2) antigen detection
1) physical behavior white or canescence Sponge Porosity agglomerate, easy and bottle wall departs from, and dissolves rapidly after adding dilution.
2) steriling test pick test, should without bacterium or fungus growth.
3) titration is randomly drawed 3 samples by different parts during freeze-drying and is carried out HA mensuration, and each sample repeats survey 2 times, and agglutination titer all should be not less than 7log2.
4) specific assay adopts the method (for details, see the appendix 1) of hemagglutination-inhibition test, respectively hemagglutination-inhibition test is carried out to H_5 subtype, H7 hypotype and H9 hypotype positive serum, newcastle disease positive serum, Egg Drop syndrome virus positive serum with this antigen, this antigen can only suppress by avian influenza virus H9 hypotype positive serum, and other positive serum to be all negative reaction.
5) residual moisture measures and is undertaken by " Chinese veterinary pharmacopoeia " residual moisture determination method, should lower than 3%.
6) vacuum tightness measures and is undertaken by " Chinese veterinary pharmacopoeia ", detects, should conform with the regulations with high-frequency electronic spark instrument.
The definite value of 4 standard substances
(1) valued methods hemagglutination test (HA test) 96 hole micro plate method measures.
(2) definite value unit has qualification by 6 ~ 8 and confirms through comparison the testing laboratory that its definite value ability is identical in advance, adopts Same Way to carry out cooperation and demarcates.
(3) definite value requirement
1) tackle each tested standard model to do respectively and independently measure for six to eight times, point two unit, each unit does three times and independently to measure; Be separated by between two unit and be no less than 3 days.6 data obtained should check exceptional value by statistical method, as found that there is exceptional value, should indicate, and do for supplement and once measure.Then whole result is quoted.
2) all measurement instrument/surveying instruments for definite value measurement need be qualified through examining and determine or calibrating, and before the deadline.
(4) data processing
1) gather whole raw data, investigate the normality of whole measurement data distribution.
2) when data Normal Distribution or approximate normal distribution, by each laboratory survey data mean value be considered as single measurements, form one group of new measurement data.Reject dubious value.Calculate population mean and standard deviation.
3) when under total data Normal Distribution or approximate normal distribution situation, one group of new measurement data is also considered as.Reject dubious value, then calculate population mean and the standard deviation of whole raw data.
5 Homogeneity Tests
(1) 25 are sampled when sample size freeze-drying quantity is more than 500; 15 are sampled when freeze-drying quantity is below 500.
(2) measurement weighs the nt wt net weight often propping up content respectively, analyzes its homogeneity with statistical method.
6 stability test antigens are preserved under-20 DEG C, 4 DEG C, 37 DEG C conditions, and regularly carry out HA valency mensuration, each time all will carry out 2 duplicate measurementss, statistically rejects dubious value with statistical method, calculate each mean value again, the deviation of this value and definite value should be not more than standard deviation.
Two, bird flu H9 hypotype blood clotting suppresses the use of antigen standard substance
Antigen system A type avian influenza virus A/Chicken/Shanghai/10/01 (H9N2) strain inoculation SPF chicken embryo, results infected chicken blastochyle, uses formalin deactivation, after concentrated, add suitable stabilizing agent freeze-drying and make.Be mainly used in:
(1), in HA test, edit H9 hypotype antigen HA and tire
The dilution of 1 standard antigen: get bird flu H9 hypotype antigen freeze-drying ampoule 1, adds 1ml physiological saline and recovers commercial weight after unpacking; First carry out 10 times, 20 times, 80 times dilutions, then carry out 1/320,1/400,1/480,1/560,1/640,1/720,1/800 times of dilution successively.
The dilution of 2 work antigens: get bird flu H9 hypotype work antigen 1 and prop up, adds physiological saline and recovers commercial weight after unpacking; First carry out 10 times, 20 times, 80 times dilutions, then carry out 320,400,480,560,640,720,800 successively ... increase progressively 80 times successively to dilute.
3 hemagglutination test (HA test)s
(1) get 96 hole V-type Microhemagglutination plates, each dilutability is got 25 μ L respectively and is joined in hole, adds physiological saline 25 μ L, then adds 1% chicken erythrocyte suspension 25 μ L.
(2) contrast: 50 μ L physiological saline+25 μ L 1% chicken erythrocyte suspensions.
(3) at micro oscillator vibration 1min ~ 2min.
(4) room temperature (24 ~ 26 DEG C) places 30min, result of determination.Blood-coagulation-board is tilted 70 ° and leave standstill and start record in about 10 seconds, have red blood cell to deposit bottom all reacting holes and the trickling in teardrop shape downwards along dip plane, present person the same as red blood cell control wells for completely not aggegation hole (-); Have red blood cell to deposit bottom reacting hole and along dip plane slowly downwards in teardrop shape trickling person for incomplete aggegation hole (+); There is red blood cell to deposit bottom reacting hole but be not 50% aggegation hole (++) along dip plane trickling person; Except a little red blood cell is arranged at bottom, other positions of reacting hole are almost complete aggegation hole person (+++); Without red blood cell bottom reacting hole, the complete aggegation person of whole reacting hole is complete aggegation hole (#).
(5) conclusion: the most high dilution corresponding when standard antigen complete aggegation hole is 460 ± 80 times, and control wells in (-) time, most high dilution corresponding to work antigen complete aggegation hole is the red cell agglutination valency of this antigen.
(2), in HI test, the HI editing avian influenza virus H9 subclass antibodies tires
The dilution of 1 antigen: get bird flu H9 hypotype antigen freeze-drying ampoule 1, adds 1ml physiological saline and recovers commercial weight after unpacking; First do 10 times, 20 times, 80 times dilutions.Then 320,400,480,560,640 times of dilutions are carried out successively.
2 hemagglutination test (HA test)s
(1) get 96 hole V-type Microhemagglutination plates, each dilutability is got 25 μ L respectively and is joined in hole, adds physiological saline 25 μ L, then adds 1% chicken erythrocyte suspension 25 μ L.
(2) contrast: 50 μ L physiological saline+25 μ L 1% chicken erythrocyte suspensions.
(3) at micro oscillator vibration 1 ~ 2min.
(4) room temperature (24 ~ 26 DEG C) is placed 30 minutes, result of determination.Blood-coagulation-board is tilted 70 ° and leave standstill and start record in about 10 seconds, have red blood cell to deposit bottom all reacting holes and the trickling in teardrop shape downwards along dip plane, present person the same as red blood cell control wells for completely not aggegation hole (-); Have red blood cell to deposit bottom reacting hole and along dip plane slowly downwards in teardrop shape trickling person for incomplete aggegation hole (+); There is red blood cell to deposit bottom reacting hole but be not 50% aggegation hole (++) along dip plane trickling person; Except a little red blood cell is arranged at bottom, other positions of reacting hole are almost complete aggegation hole person (+++); Without red blood cell bottom reacting hole, the complete aggegation person of whole reacting hole is complete aggegation hole (#).
(5) conclusion: the most high dilution that completely aggegation hole is corresponding should be 460 ± 80 times.
3 microdose cytopathogenic effect assay
(1) preparation of 4HAU antigen and checking
1) antigen diluent method
Antigen containing 400 HAUs is first carried out 10 times of dilutions, then carries out 100 times of dilutions, namely 100 times of dilutions are 4HAU hemagglutinin.The 4HAU hemagglutinin physiological saline of preparation is carried out 1: 2,1: 3,1: 4,1: 5,1: 6 and 1: 7 dilution.
2) get 96 hole V-type Microhemagglutination plates, each dilutability is got 25 μ L respectively and is joined in hole, adds physiological saline 25 μ L, then adds 1% chicken erythrocyte suspension 25 μ L.
3) contrast: 50 μ L physiological saline+25 μ L 1% chicken erythrocyte suspensions.
4) 2 parallel repetitions are done in test more than.
5) at micro oscillator vibration 1 ~ 2min.
6) room temperature (24 ~ 26 DEG C) places 30min, result of determination.
7) criterion: if preparation antigen liquid be 4HAU, then 1: 4 dilutability will provide 100% aggegation point, namely bottom reacting hole without red blood cell, the complete aggegation of whole reacting hole; If 4HAU is higher than 4 units, possibility 1: 5 or 1: 6 is 100% aggegation point; If lower, then possibility 1: 2 or 1: 3 is 100% aggegation point.Suitably should adjust according to measurement result, making antigen working fluid is really 4HAU.
(2) dilution of serum
1) standard positive serum dilute serum recover after commercial weight, carry out 10 times, 20 times and 120 times of dilutions successively; Then 720,840,960,1080 and 1200 times of dilutions are carried out.Each extension rate of the positive serum diluted is got 0.025ml, joins respectively in V-type micro-reaction plate corresponding aperture.
2) negative serum be diluted in V-type micro-reaction plate, every hole adds 0.025ml physiological saline; 1st hole adds the negative serum 0.025ml after recovering commercial weight, repeatedly lashes 3 ~ 5 mixings; Draw 0.025ml serum from the 1st hole and add the 2nd hole, draw 0.025ml after mixing and add the 3rd hole, so carry out two-fold dilution to the 6th hole, draw 0.025ml from the 6th hole and discard.
3) tested serum be diluted in V-type micro-reaction plate, every hole adds 0.025ml physiological saline; 1st hole adds the tested serum of 0.025ml, repeatedly lashes 3 ~ 5 mixings; Draw 0.025ml serum from the 1st hole and add the 2nd hole, draw 0.025ml after mixing and add the 3rd hole, so carry out two-fold dilution to the 11st hole, draw 0.025ml from the 11st hole and discard.
On 3.3V type micro-reaction plate, all serum holes add the antigen liquid 25 μ L of 4HAU.Last 1 row establishes 50 μ L physiological saline and 25 μ L physiological saline+25 μ L 4HAU antigen (each 4 holes) to contrast.
(4), after fully vibrating, under room temperature (24 ~ 26 DEG C), 30 minutes are left standstill.Every hole adds 1% chicken erythrocyte suspension 25 μ L again, fully vibrates.
(5) room temperature (24 ~ 26 DEG C) places result of determination after 30 minutes.Blood-coagulation-board is tilted 70 ° and leave standstill and start record in about 10 seconds, have red blood cell to deposit bottom all reacting holes and the trickling in teardrop shape downwards along dip plane, present person the same as red blood cell control wells for completely not aggegation hole (-); Have red blood cell to deposit bottom reacting hole and along dip plane slowly downwards in teardrop shape trickling person for incomplete aggegation hole (+); There is red blood cell to deposit bottom reacting hole but be not 50% aggegation hole (++) along dip plane trickling person; Except a little red blood cell is arranged at bottom, other positions of reacting hole are almost complete aggegation hole person (+++); Without red blood cell bottom reacting hole, the complete aggegation person of whole reacting hole is complete aggegation hole (#).
(6) conclusion
Positive serum 840 times of dilution holes are (-), and 960,1080 and 1200 times of dilution holes are (+), (++), (+++) or (#); 50 μ L saline control holes are (-), and 25 μ L physiological saline+25 μ L 4HAU antigen holes are (#), and now test can be judged to establishment.
The red cell agglutination that most high dilution corresponding to the hole of not aggegation completely of tested serum is this serum suppresses valency.
Advantage of the present invention
The present invention relates to avian influenza virus H9 hypotype blood clotting and suppress antigen standard substance and preparation method.This standard material is through the preparation of virus liquid and inspection by avian influenza virus H9 subtype C KSH01 strain virus; Virus liquid deactivation, concentrated, the inspection of semifinished product; The a series of technological process such as demarcation, definite value of freeze-drying, product inspection, Homogeneity Test and stability test and standard substance and making.This standard substance is bird flu H9 hypotype Accurate Diagnosis, immunologic surveillance and carry out the correct basic guarantee evaluated to its immune effect of vaccine, improves the prevention and control level of bird flu.
Embodiment 1
Antigen manufactures and the inspection of semifinished product
Prepared by 1 production seed culture of viruses
(1) seed culture of viruses sterile saline is made 10 times of serial dilutions by seed culture of viruses breeding, gets 10-4 dilutability, inoculation 11 age in days SPF chicken embryos in allantoic cavity, and every embryo 0.1ml, sealing pin hole, puts 37 DEG C and continue to hatch, need not egg-turning.After inoculation 24h, every 8h shines egg 1 time, and to 72h, dead chicken embryo takes out at any time, and air chamber portion is upwards upright, cools in 2 ~ 8 DEG C, and before the rear 24h of inoculation, died is discarded.The chicken embryo of cooling 8 ~ 24h is taken out, with iodine tincture disinfection air chamber position, then reject the chorion in air chamber portion with aseptic operation, throw off yolk shell membrane, break CAM and amnion (not making yolk break), sucking-off chicken blastochyle (allantoic fluid and amniotic fluid).Allantoic fluid is collected in a sterilization container.The viral seed culture of viruses of preparation should meet the standard (see annex 2 " avian influenza virus H9 hypotype hemagglutination-inhibition test antigen seed culture of viruses standard ") of this viral seed culture of viruses through inspection.
(2) preparation of virus liquid
1) production seed culture of viruses is got in inoculation, does 10 with sterile saline
-4dilution, inoculation 11 age in days SPF chicken embryos in allantoic cavity, every embryo 0.1ml, sealing pin hole, puts 37 DEG C and continues to hatch, need not egg-turning.
2) after hatching and observing egg inoculation, per sunshine egg 2 times, discarded by chicken embryo dead before 24h, chicken embryo dead after 24h takes out at any time, and to 72h, no matter whether death to be, and all take out, air chamber is upwards upright, is placed in 2 ~ 8 DEG C of cooling more than 8h.
3) the chicken embryo of cooling takes out by results, with iodine tincture disinfection air chamber position, then divest air chamber position membrana putaminis with aseptic operation, break CAM and amnion (guarding against yolk to break), push down chicken embryo with sterilizing pincet Asia, draw blastochyle with 10ml asepsis injector.Before absorption blastochyle, tackle each chicken embryo carefully check, all fetuses are corrupt, blastochyle is muddy and have the suspicious person of any pollution, are discarded.Dead germ and embryo of living are gathered in the crops respectively, and often several chicken embryos are divided into one group, draw blastochyle and are put in the neutral bottle of same sterilizing, change syringe when often changing one neutral bottle simultaneously, have often drawn one piece of embryo and Nie Zi should have been burnt on flame and be used for contacting next piece of embryo again.Preserve under the chicken blastochyle gathered in the crops is placed on 2 ~ 8 DEG C of conditions.
(3) inspection of virus liquid
1) steriling test every bottle chicken blastochyle samples respectively, and the method specified by " Chinese veterinary pharmacopoeia " is tested, should without bacterium or mould or mycoplasma growth also without exogenous virus.
2) HA valency measures every bottle of chicken blastochyle and samples respectively, sample to chicken red blood cell agglutination titer all >=7log2.
3) the above-mentioned blastochyle be up to the standards filters with sterilizing multilayer gauze by deactivation, and be mixed in a bulk container, the amount being 0.1% by ultimate density adds formalin; Preferably be poured into after adding formalin in another bottle, fail to avoid the virus adhered near bottleneck to contact inactivator.Then 37 DEG C of deactivation 24h (reach 37 DEG C with temperature in bottle and start timing, period jolting 1 time) are placed in.Preserve under blastochyle being placed in after deactivation 2 ~ 8 DEG C of conditions.
4) 10 ~ 11 age in days SPF chicken embryo 5 is got in deactivation inspection, and in each allantoic cavity, inoculation deactivation blastochyle 0.1ml, hatches 72h for 37 DEG C, results chicken blastochyle, measures coagulation for negative, and blind passage 1 generation, measure coagulation, also without blood clotting phenomenon, be judged to deactivation complete.
5) concentrated with preserve by deactivation thoroughly chicken blastochyle be dispensed in the bag filter of sterilizing, bag spreads outward and adds polyglycol, concentrated about 5 times of at room temperature dialysis.
Chicken blastochyle after concentrated is collected in sterilization container respectively, deposits under being placed in 4 ~ 8 DEG C of conditions, must not more than 3 months.
6) after inspection of semifinished product dialysis, chicken blastochyle samples respectively, carries out steriling test, without bacterium or fungus growth.After dialysis, chicken blastochyle samples respectively, and sample is to 1% chicken red blood cell agglutination titer >=9log2.
7) antigen preparation, packing and freeze-drying
Protectant preparation water for injection preparation sucrose concentration is the freeze drying protectant of 100%, for subsequent use after filtration sterilization.
Antigen prepares the chicken blastochyle that will be up to the standards, and is mixed in same container, and protective agent and chicken blastochyle are by 1: 9 proportions.
The antigen liquid ampulla prepared is carried out packing, every bottle of 1ml by packing and freeze-drying, and degree of accuracy, within ± 5%, carries out vacuum freezedrying rapidly after packing.-20 DEG C of preservations are placed after sealing.
Embodiment 2
Product inspection
1 physical behavior white or canescence Sponge Porosity agglomerate, easy and bottle wall departs from, and dissolves rapidly after adding dilution.
2 steriling test pick test, should without bacterium or fungus growth.Assay is in table 1, and all freeze-dried products are all negative.
Table 1 steriling test statistical form
3 titrations are randomly drawed sample by different parts during freeze-drying and are carried out haemagglutination mensuration (being undertaken by annex 1), and measurement result is in table 2.Result shows all to be not less than 7log2 to the agglutination titer of 1% chicken red blood cell, reaches 9log2.
Table 2 01 antigen valence testing result
Note: in table, " # " represents whole aggegation, " ++ " represents part all aggegations, and "-" represents not aggegation.
4 specific assay adopt the method (for details, see the appendix 1) of hemagglutination-inhibition tests, respectively hemagglutination-inhibition test is carried out to H_5 subtype, H7 hypotype and H9 hypotype positive serum, newcastle disease positive serum, Egg Drop syndrome virus positive serum with this antigen, this antigen can only suppress by avian influenza virus H9 hypotype positive serum, and to other positive serum all in all not suppressing.
Table 3 antigentic specificity inspection statistics table
Note: in table, " # " represents whole aggegation, namely antigen and serum do not produce and suppress to react; "-" represents not aggegation, and namely antigen and serum produce and suppresses to react.
5 residual moistures measure and are undertaken by " Chinese veterinary pharmacopoeia " residual moisture determination method, and assay is in table 4, and result shows, and the content of the residue moisture content of all samples is all less than 3%, meets the requirement of standard substance.
Table 4 remains moisture content assay
6 vacuum tightnesss measure is undertaken by " Chinese veterinary pharmacopoeia " specified vacuum degree determination method, and all ampulla vacuum tightness all meets the requirements, and occurs due aura.
Embodiment 3
The definite value that 1 standard substance is tired
(1) valued methods
Hemagglutination test (HA test) 96 hole micro plate method measures.
(2) definite value unit
By 6 ~ 8, there is qualification and confirm through comparison the testing laboratory that its definite value ability is identical in advance, adopting Same Way to carry out cooperation and demarcate.
(3) definite value requirement
1) tackle each tested standard model to do respectively and independently measure for six to 8 times, point two unit, each unit does three times and independently to measure; Be separated by between two unit and be no less than 3 days.6 data obtained should check exceptional value by statistical method, as found that there is exceptional value, should indicate, and do for supplement and once measure.Then whole result is quoted.
2) all measurement instrument/surveying instruments for definite value measurement need be qualified through examining and determine or calibrating, and before the deadline.
(4) data processing
1) gather whole raw data, investigate the normality of whole measurement data distribution.
2) when data Normal Distribution or approximate normal distribution, by each laboratory survey data mean value be considered as single measurements, form one group of new measurement data.Dubious value is rejected with statistical method.Calculate population mean and standard deviation.
3) when under total data Normal Distribution or approximate normal distribution situation, one group of new measurement data is also considered as.Reject dubious value, then calculate population mean and the standard deviation of whole raw data.
4) the definite value result cooperation calibration result of antigen valence is in table 5.Gather whole raw data, investigate the normality of whole measurement data distribution.Through descriptive statistic Epidemiological Analysis, this batch of antigen hemagglutinative titer is 460 ± 80, and final definite value is 400.
Table 5 cooperates calibration result
Note: in table, numerical value is the most highly diluted multiple of the whole aggegation of antigen.
2 Homogeneity Tests
Randomly draw 15, sample, weigh its nt wt net weight respectively with electronic analytical balance, the results are shown in Table 2-6.Descriptive biometric analysis draws: example weight is all between 0.112 ~ 0.13g, and average is 0.123g; CV is 4.1%, all in 95% range of normal value; Result shows that this batch of standard substance loading amount is homogeneous.
Table 6 sample weighing analysis result
3 stability tests
The results are shown in Table 7,8,9, result shows that this antigen is preserved 28 days HA valencys and do not declined under 37 DEG C of conditions, within 35 days, slightly reduces.Preserve HA valency when preserving 18 months for 6 months and-20 DEG C for 4 DEG C not decline.
Table 7 stability test result (one)
Table 8 stability test result (two)
Table 9 stability test result (three)
Annex 1
Bird flu blood clotting (HA) and blood clotting suppress (HI) test
1 material
1.1 96 hole V-types (90 degree) micro-reaction plate, single track and multichannel micropipettor (being furnished with suction nozzle), loading slot, suction pipe, beaker etc.
1.2 0.85% physiological saline
1.2.1 normal saline measures 8.5g NaCl, and adding distil water is to 1000ml;
1.2.2 with 121 DEG C of sterilizings 30 minutes, for subsequent use;
1.2.3 physiological saline is once use, is no more than 1 week in 2 ~ 8 DEG C of preservations.
1.3 A Shi (Alsevers) liquid claims glucose 2.05g, sodium citrate 0.8g, citric acid 0.055g, sodium chloride 0.42g, and adding distil water is to 100ml, and adjust pH to 6.1 after heating for dissolving, 69Kpa 15min autoclaving, 2 ~ 8 DEG C save backup.
1.4 1% chicken erythrocyte suspensions gather 2 ~ 3 SPF cocks or mix with equal-volume A Shi liquid without the blood of the healthy cock of the antibody such as bird flu and ewcastle disease, with 0.85% brine 3 times, at every turn all with the centrifugal 5min of 3000rpm/min, be made into 1% (V/V) red cell suspension with physiological saline after washing, 2 ~ 8 DEG C save backup.
The antigen of 1.5 antigens dissolving freeze-drying and serum, all by the amount of specification mark on label, use physiological saline solution.
2 operation art formulas
2.1 blood clottings (HA) are tested
2.1.1, in V-type micro-reaction plate, every hole adds 0.025ml physiological saline.
2.1.2 the 1st hole adds 0.025ml antigen, repeatedly lashes 3 ~ 5 mixings.
2.1.3 draw 0.025ml antigen from the 1st hole and add the 2nd hole, draw 0.025ml after mixing and add the 3rd hole, so carry out two-fold dilution to the 11st hole, draw 0.025ml from the 11st hole and discard.
2.1.4 every hole adds 0.025ml physiological saline.
2.1.5 every hole adds 0.025ml 1% (V/V) chicken erythrocyte suspension.
2.1.6 reaction plate is shaken on the oscillator 1 ~ 2 minute or light button reaction plate mixed reactant, under room temperature (20 ~ 25 DEG C), leave standstill 20 ~ 30 minutes or 2 ~ 8 DEG C 45 ~ 60 minutes.The result of determination when control wells red blood cell is significantly in button-type.
2.1.7 result judges reaction plate to tilt 60 degree, observes red blood cell and trickles with or without teardrop shape, is completely hemagglutinative titer without the trickle most highly diluted multiple of (100% aggegation) of teardrop sample.
2.2 blood clottings suppress (HI) test
Tiring 2.2.1 according to HA test determination, calculates preparation 4 HAU (4HAU) antigens.HA tires and is extension rate containing 4HAU antigen divided by 4.Such as, it is 1: 256 that HA tires, then the extension rate of 4HAU antigen should be 1: 64 (256 divided by 4).
2.2.2 1st ~ 11 holes add 0.025ml physiological saline, and the 12nd hole adds 0.05ml physiological saline.
2.2.3 the 1st hole adds 0025ml serum, and fully inhale 0.025ml after mixing in the 2nd hole, two-fold dilution is to the 10th hole successively, draws 0.025ml discard from the 10th hole.
2.2.4 1st ~ 11 holes all add the 4HAU antigen of 0.025ml, under room temperature (20 ~ 25 DEG C) leave standstill 30 minutes or 2 ~ 8 DEG C 50 minutes.
2.2.5 every hole adds the chicken erythrocyte suspension of 0.025ml 1% (V/V), concussion mixing, under room temperature (20 ~ 25 DEG C), leave standstill 20 ~ 30 minutes or 2 ~ 8 DEG C 45 ~ 60 minutes, contrast red blood cell will be sunken at the bottom of hole in obvious button-type.
3 results judge to tire with the HI suppressing the highest serum extension rate of 4HAU antigen to be judged to this serum completely.Be no more than 1 titre when the HI of positive control serum tires with known error of tiring, when negative control sera is tired not higher than 2log2, test can be set up.Tested serum HI tires≤and 3log2 is judged to feminine gender;=4log2 be judged to suspicious (suspicious specimen should heavily be examined, heavily inspection tire >=4log2 is judged to the positive ,≤3log2 is judged to feminine gender); >=5log2 is judged to the positive.
Annex 2
Avian influenza virus H9 hypotype hemagglutination-inhibition test antigen seed culture of viruses standard
1 red cell agglutination valency answers >=8log2 to 1% chicken red blood cell agglutination titer.
Seed culture of viruses sterile saline is made 10 times of serial dilutions by 2 viral levels, gets 10-6,10-7,10-8 3 dilutabilitys, inoculation 10 age in days SPF chicken embryo 5 in each allantoic cavity, every embryo 0.1ml.Put 37 DEG C to continue to hatch, chicken embryo dead before 24 hours discards to be disregarded, and chicken embryo dead in 24 ~ 72 hours takes out and at any time in 2 ~ 8 DEG C of stored refrigerated.To 72 hours, measure the red cell agglutination valency of all chicken blastochyles, agglutination titer >=4log2 person was judged to infection, calculated EID50, and every ml viral level answers >=107.5EID50.
3 specificity micromethods carry out hemagglutination inhibition test (HI test), should be positive reaction, all should be negative reaction to H5 and H7 subtype avian influenza, newcastle disease and Egg Drop syndrome virus positive serum to avian influenza virus H9 hypotype antiserum.
Seed culture of viruses sterile saline is done 10 times of dilutions by the virulence of 4 pairs of chickens, collunarium inoculation SPF chicken in 4 ~ 5 week age 10, every 0.1ml.The SPF chicken 5 that condition of separately getting is identical, does not inoculate in contrast, raises respectively under same condition, observes 14, and infected chicken and contrast chicken all should not occur any abnormal response.
5 pure press " Chinese veterinary pharmacopoeias " carry out, and should pollute without bacterium, mould, mycoplasma and exogenous virus.
Within 6 basic bacteria algebraically 5 generations.
7 seeds culture of viruses are kept at less than-70 DEG C, and storage life is 60 months.
Claims (3)
1. an avian influenza virus H9 hypotype hemagglutination-inhibition test antigen standard substance, is characterized in that this antigen standard substance contains avian influenza virus H9 hypotype A/Chicken/Shanghai/10/01 (H9N2) strain virus of deactivation; Hemagglutination-inhibition test antigen to chicken red blood cell agglutination titer all >=7log2; This antigen can only suppress by avian influenza virus H9 hypotype positive serum, and to H_5 subtype, H7 hypotype positive serum, newcastle disease positive serum and Egg Drop syndrome virus positive serum are all negative reaction.
2. a preparation method for avian influenza virus H9 hypotype hemagglutination-inhibition test antigen standard substance, it is characterized in that by utilizing by avian influenza virus H9 hypotype A/Chicken/Shanghai/10/01 (H9N2) strain virus of isolated in China through following steps:
(1) avian influenza virus H9 hypotype hemagglutination-inhibition test antigen standard substance material standed for is prepared:
The preparation of H9 subtype avian influenza virus seed culture of viruses and inspection;
The preparation of virus liquid and inspection;
Virus liquid deactivation, concentrated, the inspection of semifinished product;
Freeze-drying, product inspection, Homogeneity Test and stability test;
(2) demarcation of standard substance, definite value.
3. the preparation method of a kind of avian influenza virus H9 hypotype hemagglutination-inhibition test antigen standard substance as claimed in claim 2; it is characterized in that the freeze drying protectant used in preparation process is: preparing sucrose concentration with water for injection is the freeze drying protectant of 100%; after filtration sterilization, protective agent and chicken blastochyle press 1:9 proportions.
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| 禽流感病毒人工感染鸡抗体的监测及血凝素分型血清的制备;田国斌等;《中国畜禽传染病》;19980101;第20卷(第1期);第32-34页 * |
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