CN102012430B - Avian influenza H5N1 subtype Re-5 strain hemagglutination inhibition antigen standard substance and preparation method - Google Patents
Avian influenza H5N1 subtype Re-5 strain hemagglutination inhibition antigen standard substance and preparation method Download PDFInfo
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Abstract
The invention relates to an avian influenza H5N1 subtype Re-5 strain hemagglutination inhibition antigen standard substance and a preparation method thereof. The standard substance is prepared by performing the following series of steps of: preparing and inspecting liquid of avian influenza H5N1 subtype Re-5 strain viruses; inactivating the liquid of the viruses and inspecting a semi-finished product; freeze-drying, inspecting a finished product, homogeneity and stability, demarcating the standard substance, valuing and the like. The standard substance is fundamental guarantee of accurately diagnosing avian influenza H5N1 subtype, performing immune monitoring of an H5N1 subtype Re-5 strain hemagglutination inhibition antibody and accurately evaluating the immune effect of vaccine and improves the prevention and control level of the avian influenza.
Description
Technical field
The present invention relates to a kind of bird flue virus H 5 N 1 subtype Re-5 strain blood clotting and suppress antigen standard substance and preparation method, belong to the veterinary biologics field.
Background technology
The chemistry contrast material of British Pharmacopoeia (BP) is just to propose since 1963, because WHO has set up international biological standard material center in Britain, so Britain continues to use international reference materials always, and its national standard substance is set up later, after 1970, European Pharmacopoeia comes out, and Britain is except quoting international reference materials, also use the European Pharmacopoeia standard substance, a small amount of standard substance that uses this country.Even so, BP1968 version and enlarged edition thereof add the quantity that British Pharmacopoeia (nineteen sixty-eight) records again and surpass 260, and version surpassed 300 in 1980, and the BP1993 version has been recorded 371.The American Pharmacopeia council can provide about more than 1805 kinds of drug standards materials; European Pharmacopoeia Commission has 2098 kinds, and wherein the biological product standards material has 319 kinds.
At present, China has developed 1262 kinds of country-level standard substances, and 1739 kinds of secondary reference materials relate to fields such as iron and steel, geology, oil, nuclear material, environment, food and clinical examination.The standard substance research of veterinary biologics lags behind, and have only 192 kinds at present, and the veterinary biologics check is almost nil with standard substance.
Whether accurately the veterinary biologics standard substance is control and definite veterinary biologics product quality, calibration verification testing instruments and method standard of physical, is correctly to diagnose infectious disease, accurately monitor the material base of immune situation.
Bird flu (AI) is the great animal epidemic of China's emphasis prevention and control, and avian influenza virus (AIV) H5 hypotype belongs to highly pathogenic virus, and H5N1 hypotype Re-5 strain is developed at the HPAIV of China's current popular.Inactivated avian influenza vaccine with this strain development is most widely used first-selected vaccine in the anti-system of the present bird flu of China, and this vaccine has been obtained very significant immune effect in actual applications, and therefore the anti-manufacture-illegal of the bird flu of China in recent years is often satisfactory.Most widely used method is the HI test in diagnosis, immunologic surveillance and the immune effect of vaccine evaluation thereof of AIV H5N1 hypotype at present, this is the method that China Ministry of Agriculture in 2006 approves, but China does not still have the report that standard substance and correlative study thereof are used in the hemagglutination-inhibition test of AIV H5 hypotype at present, external International Animal Health tissue reference laboratory such as (OIE) does not have yet, and different times detects the consistance of data and the comparability of different experiments number of chambers certificate before and after having had a strong impact on.In recent years laboratories at different levels are more and more to the demand of AIV standard substance, the diagnostic reagent standard substance that particularly the H5 hypotype is relevant with the H9 hypotype, therefore it is particularly urgent to develop AIVH5 hypotype blood clotting inhibition antigen standard substance, this standard substance is that AIV H5 hypotype is accurately diagnosed, AIV H5N1 hypotype Re-5 strain immunity condition monitoring is reached the basic guarantee that its immune effect of vaccine is correctly estimated, and improves the prevention and control level of bird flu.
The development main reference of present China standard substance 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, but this technical manual is the development that is applicable to chemical constitution, physicochemical characteristics and engineering characteristic primary standard material, for the biological product standards material, particularly veterinary biologics standard substance development does not still have technical manual at present and can follow.Therefore the development of veterinary biologics standard substance is a footless research.
Summary of the invention
The present invention suppresses the antigen standard substance for a kind of bird flue virus H 5 N 1 subtype Re-5 strain blood clotting of preparation, and this antigen standard substance contains the bird flue virus H 5 N 1 subtype Re-5 strain virus of deactivation; Hemagglutination-inhibition test antigen to the chicken red blood cell agglutination titer all 〉=8log2; This antigen can only be suppressed by avian influenza virus H5 hypotype Re-5 strain positive serum, and to avian influenza virus H9 hypotype, H7 hypotype positive serum, the reaction that all is negative of newcastle disease positive serum and egg drop syndrome virus-positive serum.
Main preparation methods is: (1) preparation bird flue virus H 5 N 1 subtype Re-5 strain virus hemagglutination-inhibition test antigen standard substance material standed for: preparation and the check of H5N1 hypotype Re-5 strain avian influenza virus seed culture of viruses; Preparation and the check of virus liquid; Beta-propiolactone deactivation, the inspection of semifinished product of virus liquid; Freeze-drying, product inspection, homogeneity check and stability test;
(2) demarcation of standard substance, definite value.
Elaborating of technical solution of the present invention
Comprise contents such as bird flue virus H 5 N 1 subtype Re-5 strain hemagglutination-inhibition test antigen standard substance, its preparation method and program, its preparation rules and cooperation proving operation standard.
The inventor is on the existing AIVH5N1 hypotype Re-5 strain blood clotting inhibition method basis of country, having formulated bird flue virus H 5 N 1 subtype Re-5 strain blood clotting suppresses the quality standard of antigen standard substance and prepares rules, developed bird flue virus H 5 N 1 subtype Re-5 strain blood clotting and suppressed the antigen standard substance, and with 8 tame units such as China Veterinery Drug Inspection Office, China Agricultural University it has been tired and to have carried out the demarcation that cooperates.
One, bird flue virus H 5 N 1 subtype Re-5 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 for reorganization bird flue virus H 5 N 1 subtype Re-5 strain for the manufacture of bird flue virus H 5 N 1 subtype Re-5 strain blood clotting, identify, take care of and supply by China national bird flu reference laboratory (in Chinese Harbin City Harbin Veterinary Medicine Inst., China Academy of Agriculture).
(2) characteristic of viral seed culture of viruses
1) HA tires and carries out the chicken red blood cell determination of agglutination titer by the method for this instructions appendix (being called for short " appendix "), and Re-5 strain seed culture of viruses is answered 〉=9log2 1% chicken red blood cell agglutination titer.
2) chicken embryo median infective dose is made 10 times of serial dilutions with Re-5 strain seed culture of viruses with sterile saline, gets 10
-6, 10
-7, 10
-8Inoculation 10 age in days SPF chicken embryos are 5 in 3 dilutabilitys, each allantoic cavity, every embryo 0.1ml.Put 37 ℃ and continue to hatch, dead chicken embryo discards and disregards before the 24h, and dead chicken embryo takes out and at any time in 2～8 ℃ of stored refrigerated in 24～72h.To 72h, measure the red cell agglutination valency (" appendix ") of all chicken blastochyles, agglutination titer 〉=4log2 person is judged to infection, calculates EID
50(the Chinese veterinary drug allusion quotation council. in 2005 version (three ones) of People's Republic of China's veterinary drug allusion quotation. Chinese agriculture publishing house, 2006, the present invention is called for short " Chinese veterinary drug allusion quotation "), every ml viral level should 〉=10
7.0EID
50
3) identified for genes method and standard are got Re-5 strain seed culture of viruses 200 μ l, extract kit (QIAGEN company) with QIAGEN RNA and extract viral RNA, with upstream primer ACATAGTGAGAAGAGCTG and downstream primer TCCCACTCACTATCAGCTG, adopt the genetic fragment of RT-PCR method amplification HA cracking site, amplification PCR products is carried out sequential analysis.AIV Re-5 strain HA gene cracking site amino acid sequence should be-RETR ↓-, meet typical low pathogenicity feature.
4) virulence of chicken embryo is made 10 times of serial dilutions to 10 with Re-5 strain seed culture of viruses with sterile saline
-4, with 10 piece of 10 age in days SPF chick embryo allantoic cavity of this concentration virus liquid inoculation, every embryo 0.1ml puts 36 ℃ and hatches 72h, and is should at least 9 chicken embryos not dead, and idiosome does not have the visible pathology of naked eyes.
5) virulence of chick is done 10 times of dilutions with Re-5 strain seed culture of viruses with sterile saline, 10 of intravenous inoculation SPF chickens in 5～6 age in week, every 0.1ml, intravenous inoculation pathogenic index are 0; 10 of collunarium inoculation SPF chickens in 3～4 age in week, every 0.1ml (contains 10
6EID
50), other gets 10 of the identical SPF chickens of condition, does not inoculate in contrast, with raising respectively under the condition, observes 14, death or obviously abnormal response all should not occur.
6) specificity is carried out the HI test with micromethod, Re-5 strain seed culture of viruses should be positive reaction to avian influenza virus H5 hypotype antiserum, and avian influenza virus H1～H4, H6～H15 hypotype, newcastle disease virus and egg drop syndrome virus monospecific antiserum all should negatively be reacted.
7) pure property should not have bacterium, mould, mycoplasma and exogenous virus) pollution (testing by " Chinese veterinary drug allusion quotation " prescriptive procedure).
8) in 2～5 generations of subalgebra, are planted on the basis.
9) seed culture of viruses is kept at below-70 ℃, and storage life is 60 months.
2. animal used as test
Employed SPF chicken and SPF chicken embryo provide by Harbin veterinary institute Experimental Animal Center.
3. bird flue virus H 5 N 1 subtype Re-5 strain blood clotting suppresses the preparation of antigen standard substance material standed for
(1) preparation of antigen
According to conventional method, seed culture of viruses is inoculated SPF chicken embryo, results chicken blastochyle prepares bird flue virus H 5 N 1 subtype Re-5 strain blood clotting and suppresses the antigen semi-manufacture, through the beta-propiolactone deactivation, concentrate, carry out steriling test, titration after, by every bottle of 1ml packing freeze-drying.Protective agent adopts the prescription of this problem development and compound method (be 100% freeze drying protectant with water for injection preparation sucrose concentration, after the filtration sterilization, protective agent and chicken blastochyle are prepared in 1: 9 ratio).
(2) antigen check
1) physical behavior antigen is the spongy loose agglomerate of white or canescence, easily breaks away from the bottle wall, adds dissolving rapidly behind the dilution.
2) steriling test pick test should not have bacterium or fungus growth.
Different parts was randomly drawed 3 samples and is carried out HA and measure when 3) titration was by freeze-drying, and each sample repeats to survey 2 times, and agglutination titer should all be not less than 8log2.
4) method (seeing " appendix " for details) of hemagglutination-inhibition test is adopted in the specificity check, respectively avian influenza virus H5 hypotype, H7 hypotype and H9 hypotype positive serum, newcastle disease positive serum, egg drop syndrome virus-positive serum are carried out hemagglutination-inhibition test with this antigen, this antigen can only be suppressed by bird flue virus H 5 N 1 subtype (Re-5 strain) positive serum, and to the reaction that all is negative of other positive serum.
5) residual moisture is measured by " Chinese veterinary drug allusion quotation " residual moisture determination method and is undertaken, and should be lower than 3%.
6) vacuum tightness is measured by " Chinese veterinary drug allusion quotation " method and is undertaken, and detects with high-frequency electronic spark instrument, should be up to specification.
The definite value of 4 standard substances
(1) valued methods hemagglutination test (HA test) 96 hole micro plate methods are measured
(2) definite value unit is had qualification and is in advance confirmed the identical testing laboratory of its definite value ability through comparison by 6～8 families, adopts the Same Way demarcation that cooperates.
(3) definite value requirement
1) tackles each tested standard model and do independently mensuration respectively 6～8 times, measure and divide two unit, each unit to do independent mensuration three times; Be separated by between two unit and be no less than 3 days.6 data that obtain should be checked exceptional value by statistical method, as finding exceptional value are arranged, and should indicate, and do for supplement once and measure.Quote whole results then.
2) measurement instrument/surveying instrument of measuring of the definite value that is useful on need through calibrating or calibrate qualified, and before the deadline.
(4) data are handled
1) gathers whole raw data, investigate the normality that whole measurement data distribute.
2) under the situation that data Normal Distribution or similar normal state distribute, the mean value of the data of surveying in each laboratory is considered as the single measurement value, constitute one group of new measurement data.Reject dubious value.Calculate population mean and standard deviation.
3) under total data Normal Distribution or similar normal state distribution situation, also be considered as one group of new measurement data.Reject dubious value, calculate population mean and the standard deviation of whole raw data again.
5. homogeneity check
(1) sample size freeze-drying quantity is 25 of 500 above time samplings; Freeze-drying quantity is 15 of 500 following time samplings.
(2) measure the nt wt net weight of every content of weighing respectively, analyze its homogeneity with statistical method.
6. stability test antigen is preserved under-20 ℃, 4 ℃, 37 ℃ conditions, and regularly carry out the HA valency and measure, each time all will be carried 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, H 5 N 1 avian influenza hypotype Re-5 strain blood clotting suppresses the use of antigen standard substance
Antigen system gathers in the crops the infected chicken blastochyle with recombinant fowl influenza virus H5N1 hypotype Re-5 strain inoculation SPF chicken embryo, uses the beta-propiolactone deactivation, after concentrating, adds suitable stabilizing agent freeze-drying and makes.Be mainly used in:
(1) in the HA test, edits H5N1 hypotype Re-5 strain antigen HA and tire
1. 1 of H 5 N 1 avian influenza hypotype Re-5 strain antigen freeze-drying ampoule is got in the dilution of standard antigen, adds 1ml physiological saline after the unpacking and recovers commercial weight; Carry out 10 times, 20 times dilutions earlier, carry out 80,160,320,400,480,560 times of dilutions then successively.
2. the dilution of work antigen is got bird flu H9 hypotype work antigen 1 and is propped up, and adds physiological saline after the unpacking and recovers commercial weight; Carry out 10 times, 20 times dilutions earlier, carry out 80,160,320,400,480,560,640,720,800 then ... increasing progressively 80 times successively dilutes.
3. hemagglutination test (HA test)
(1) get 96 hole V-type Microhemagglutination plates, each dilutability is got 25 μ L respectively and is joined in the hole, adds physiological saline 25 μ L, adds 1% chicken erythrocyte suspension, 25 μ L again.
(2) contrast: 50 μ L physiological saline+25 μ L, 1% chicken erythrocyte suspension.
(3) at micro oscillator vibration 1min～2min.
(4) room temperature (24～26 ℃) is placed 30min, result of determination.Blood-coagulation-board is tilted 70 ° and leave standstill 10 seconds left and right sides opening entries, and there is the red blood cell deposition all reacting holes bottom and is the teardrop shape trickling downwards along the dip plane, presents the same person with the red blood cell control wells and is not aggegation hole (-) fully; There is the red blood cell deposition reacting hole bottom and slowly is teardrop shape trickling person downwards along the dip plane is incomplete aggegation hole (+); There is the red blood cell deposition reacting hole bottom but is not 50% aggegation hole (++) along dip plane trickling person; Except a little red blood cell was arranged at the bottom, other positions of reacting hole were almost complete aggegation hole person (+++); The no red blood cell in reacting hole bottom, the complete aggegation person in entire reaction hole is complete aggegation hole (#).
(5) conclusion when the high dilution of the complete aggegation of standard antigen hole correspondence be 300 ± 60 times, and control wells is when being (-), the high dilution of the complete aggegation of work antigen hole correspondence is the red cell agglutination valency of this antigen.
(2) in the HI test, the HI that edits bird flue virus H 5 N 1 subtype (Re-5 strain) antibody tires
1. the dilution of antigen: get 1 of H 5 N 1 avian influenza hypotype Re-5 strain antigen freeze-drying ampoule, add 1ml physiological saline after the unpacking and recover commercial weight; Do 10 times, 20 times dilutions earlier.Carry out 80,160,320,400,480,560 times of dilutions then successively.
2. hemagglutination test (HA test)
(1) get 96 hole V-type Microhemagglutination plates, each dilutability is got 25 μ L respectively and is joined in the hole, adds physiological saline 25 μ L, adds 1% chicken erythrocyte suspension, 25 μ L again.
(2) contrast: 50 μ L physiological saline+25 μ L1% chicken erythrocyte suspensions.
(3) at micro oscillator vibration 1～2min.
(4) room temperature (24～26 ℃) is placed 30min, result of determination.Blood-coagulation-board is tilted 70 ° and leave standstill 10 seconds left and right sides opening entries, and there is the red blood cell deposition all reacting holes bottom and is the teardrop shape trickling downwards along the dip plane, presents the same person with the red blood cell control wells and is not aggegation hole (-) fully; There is the red blood cell deposition reacting hole bottom and slowly is teardrop shape trickling person downwards along the dip plane is incomplete aggegation hole (+); There is the red blood cell deposition reacting hole bottom but is not 50% aggegation hole (++) along dip plane trickling person; Except a little red blood cell was arranged at the bottom, other positions of reacting hole were almost complete aggegation hole person (+++); The no red blood cell in reacting hole bottom, the complete aggegation person in entire reaction hole is complete aggegation hole (#).
(5) conclusion: fully the high dilution of aggegation hole correspondence should be 300 ± 60 times.
3. Microhemagglutination inhibition test
(1) preparation of 4HAU antigen and checking
1) antigen diluent method
The antigen that will contain 320 HAUs carries out 10 times of dilutions earlier, carries out 8 times of dilutions again, and namely 80 times of dilutions are the 4HAU hemagglutinin.The 4HAU hemagglutinin of preparation was carried out 1: 2,1: 3,1: 4,1: 5,1: 6 and dilution in 1: 7 with physiological saline.
2) get 96 hole V-type Microhemagglutination plates, each dilutability is got 25 μ L respectively and is joined in the hole, adds physiological saline 25 μ L, adds 1% chicken erythrocyte suspension, 25 μ L again.
3) contrast: 50 μ L physiological saline+25 μ L, 1% chicken erythrocyte suspension.
4) 2 parallel repetitions are done in above test.
5) at micro oscillator vibration 1～2min.
6) room temperature (24～26 ℃) is placed 30min, result of determination.
7) criterion: if the antigen liquid of preparation is 4HAU, then 1: 4 dilutability will provide 100% aggegation, i.e. the no red blood cell in reacting hole bottom, the complete aggegation in entire reaction hole; If 4HAU is higher than 4 units, possibility was 100% aggegation point in 1: 5 or 1: 6; If lower, then possibility was 100% aggegation in 1: 2 or 1: 3.Should suitably adjust according to measurement result, make the antigen working fluid really be 4HAU.
(2) dilution of serum
1) standard positive serum dilute serum recover commercial weight after, carry out 10 times, 20 times dilutions successively; Carry out 80,160,240,320,400,480,560 times of dilutions then.Each extension rate of positive serum that dilution is good is got 0.025ml, joins respectively in the V-type micro-reaction plate corresponding aperture.
2) being diluted in the V-type micro-reaction plate of negative serum, every hole adds 0.025ml physiological saline; The 1st hole adds the negative serum 0.025ml after the recovery commercial weight, lashes mixing repeatedly 3～5 times; Draw 0.025ml serum from the 1st hole and add the 2nd hole, draw 0.025ml behind the mixing and add the 3rd hole, so carry out two-fold dilution to the 6 holes, draw 0.025ml from the 6th hole and discard.
3) dilution of tested serum
Serum carries out 10 times, 20 times dilutions after recovering commercial weight successively; Carry out 80,160,240,320,400,480,560 then ... 120 times increase progressively and dilute successively.To dilute each extension rate of serum and get 0.025ml, join respectively in the V-type micro-reaction plate corresponding aperture.
(4) after the abundant vibration, leave standstill 30min under the room temperature (24～26 ℃).Every hole adds 1% chicken erythrocyte suspension, 25 μ L again, fully vibration.
(5) result of determination behind room temperature (24～26 ℃) the placement 30min.Blood-coagulation-board is tilted 70 ° and leave standstill 10 seconds left and right sides opening entries, and there is the red blood cell deposition all reacting holes bottom and is the teardrop shape trickling downwards along the dip plane, presents the same person with the red blood cell control wells and is not aggegation hole (-) fully; There is the red blood cell deposition reacting hole bottom and slowly is teardrop shape trickling person downwards along the dip plane is incomplete aggegation hole (+); There is the red blood cell deposition reacting hole bottom but is not 50% aggegation hole (++) along dip plane trickling person; Except a little red blood cell was arranged at the bottom, other positions of reacting hole were almost complete aggegation hole person (+++); The no red blood cell in reacting hole bottom, the complete aggegation person in entire reaction hole is complete aggegation hole (#).
(6) conclusion
It is 200 ± 40 times that positive serum HI tires; 50 μ L physiological saline control wells are (-), and 25 μ L physiological saline+25 μ L4HAU antigen holes are (#), and this moment, test can be judged to establishment.
The red cell agglutination that the high dilution of the hole of the not aggegation fully correspondence of tested serum is this serum suppresses valency.
Advantage of the present invention
The present invention relates to bird flue virus H 5 N 1 subtype Re-5 strain blood clotting and suppress antigen standard substance and preparation method.This standard substance is by preparation and the check of bird flue virus H 5 N 1 subtype Re-5 strain virus through viral liquid; The deactivation of virus liquid, the inspection of semifinished product; A series of technological processs such as the demarcation of freeze-drying, product inspection, homogeneity check and stability test and standard substance, definite value and making.This standard substance is that the H 5 N 1 avian influenza hypotype is accurately diagnosed, H 5 N 1 avian influenza hypotype Re-5 strain immunologic surveillance reaches the basic guarantee that its immune effect of vaccine is correctly estimated, and improves the prevention and control level of bird flu.
Embodiment 1
Antigen is made and the inspection of semifinished product
1 production prepares with seed culture of viruses
(1) seed culture of viruses breeding: seed culture of viruses is made 10 times of serial dilutions with sterile saline, get 10
-4Inoculation 10 age in days SPF chicken embryos in the dilutability, allantoic cavity, every embryo 0.1ml, the sealing pin hole is put 36 ℃ and is continued to hatch, needn't egg-turning.Behind the inoculation 24h, every 12h is according to egg 1 time, dead chicken embryo take out at any time discard need not, collect all embryos of living, cooling in 2～8 ℃ to 72h.The chicken embryo of cooling 4～24h is taken out, with tincture of iodine sterilization air chamber position, reject the chorion of air chamber portion then with aseptic operation, throw off the yolk shell membrane, break CAM and amnion (yolk is broken), sucking-off chicken blastochyle (allantoic fluid and amniotic fluid).Allantoic fluid is collected in respectively in the sterilization container.Carry out steriling test and HA valency one by one and measure, will check aseptic and the chicken blastochyle of 1% chicken erythrocyte suspension agglutination titer 〉=10log2 be mixed, quantitatively packing, harvest date, seed culture of viruses generation etc. are indicated in-70 ℃ of preservations.
(2) preparation of viral liquid
1) inoculation is got to produce and is used seed culture of viruses, makes 10 times of serial dilutions to 10 with sterile saline
-4, inoculation 11 age in days SPF chicken embryos in the allantoic cavity, every embryo 0.1ml, the sealing pin hole is put 37 ℃ and is continued to hatch, needn't egg-turning.
2) hatch and observe egg inoculation after, discard 24h before dead chicken embryo according to egg 2 times every day, chicken embryo dead behind the 24h takes out at any time, to 72h, no matter whether death to be, all takes out, air chamber is upwards upright, places more than 2～8 ℃ of cooling 8h.
3) results are taken out the chicken embryo of cooling, with tincture of iodine sterilization air chamber position, divest air chamber position membrana putaminis with aseptic operation then, break CAM and amnion (guarding against yolk breaks), push down the chicken embryo with sterilization pincet Asia, draw blastochyle with the 10ml asepsis injector.Each chicken embryo scrutiny of reply before drawing blastochyle, all fetus corruption, blastochyle is muddy and the suspicious person of any pollution is arranged, and discarding need not.Dead germ and the embryo of living are gathered in the crops respectively, and whenever several chicken embryos are divided into one group, draw blastochyle and are put in the neutral bottle of same sterilization, change syringe during one neutral bottle of every replacing simultaneously, and every absorption finishes one piece of embryo and Nie Zi should be burnt at flame and be used for contacting next piece embryo again.The chicken blastochyle of results is placed under 2～8 ℃ of conditions preserves.
(3) check of viral liquid
1) every bottle of chicken blastochyle of steriling test is taken a sample respectively, tests by the method for " Chinese veterinary drug allusion quotation " regulation, and no bacterium or mould or mycoplasma growth do not have exogenous virus yet.
2) every bottle of chicken blastochyle of HA valency mensuration is taken a sample respectively, and sample is not less than 9log2 to the chicken red blood cell agglutination titer.
3) deactivation filters the above-mentioned blastochyle that is up to the standards with sterilization multilayer gauze, be mixed in the bulk container, and be that 0.03% amount adds beta-propiolactone by ultimate density, abundant mixing places to take out behind 4 ℃ of deactivation 24h to place 37 ℃ of water-baths to stop deactivation 1h again.After the deactivation blastochyle placed under 2～8 ℃ of conditions and preserve.
4) 5 of 10～11 age in days SPF chicken embryos are got in the deactivation check, and inoculation deactivation blastochyle 0.1ml is hatched 72h for 37 ℃ in each allantoic cavity, results chicken blastochyle, measure blood clotting (seeing " appendix " for details), negative, and 1 generation of blind passage, measure blood clotting, no blood clotting phenomenon, it is complete to be judged to deactivation.
5) inspection of semifinished product chicken blastochyle is taken a sample respectively, carries out steriling test, no bacterium or fungus growth.The chicken blastochyle is taken a sample respectively, and sample is to 1% chicken red blood cell agglutination titer 〉=9log2.
7) antigen preparation, packing and freeze-drying
Protectant preparation is 100% freeze drying protectant with water for injection preparation sucrose concentration, and is standby after the filtration sterilization.
Antigen preparation is sneaked into the chicken blastochyle that is up to the standards in the same container, and protective agent and chicken blastochyle are prepared in 1: 9 ratio.
Packing and freeze-drying are carried out packing with the antigen liquid for preparing with the peace bottle, every bottle of 1ml, and degree of accuracy is carried out vacuum freezedrying rapidly after the packing in ± 5%.-20 ℃ of preservations are placed in the sealing back.
Embodiment 2
Product inspection
1. the spongy loose agglomerate of physical behavior white or canescence easily breaks away from the bottle wall, adds dissolving rapidly behind the dilution.
2. steriling test pick test, no bacterium or fungus growth.Assay sees Table 1, and all freeze-dried products all are negative.
Table 1 steriling test statistical form
3. different parts was randomly drawed sample and is carried out haemagglutination mensuration (being undertaken by " appendix ") when titration was by freeze-drying, and measurement result sees Table 2.The result shows that the agglutination titer to 1% chicken red blood cell is 8log2.
Table 201 antigen valence testing result
Annotate: " # " represents whole aggegations in the table, all aggegations of " ++ " expression part, and "-" represents not aggegation.
4. the method (seeing " appendix " for details) of hemagglutination-inhibition test is adopted in the specificity check, respectively avian influenza virus H5 hypotype, H7 hypotype and H9 hypotype positive serum, newcastle disease positive serum, egg drop syndrome virus-positive serum are carried out hemagglutination-inhibition test with this antigen, this antigen can only be suppressed by the positive serum of avian influenza virus H5 hypotype Rel strain, and other positive serum is not all suppressed.
Table 3 antigentic specificity inspection statistics table
Annotate: " # " represents whole aggegations in the table, and namely antigen and serum do not produce inhibitory reaction; "-" represents not aggegation, and namely antigen and serum produce inhibitory reaction.
5. residual moisture is measured by " Chinese veterinary drug allusion quotation " residual moisture determination method and is undertaken, and assay sees Table 4, and the result shows that the content of the residue moisture content of all samples meets the requirement of standard substance all less than 3%.
Table 4 residue moisture content assay
6. vacuum tightness is measured by " Chinese veterinary drug allusion quotation " specified vacuum degree determination method and is undertaken, and all peace bottle vacuum tightnesss all meet the requirements, and due aura occurs.
Embodiment 3
1. the standard substance definite value of tiring
(1) valued methods
Hemagglutination test (HA test) 96 hole micro plate methods are measured.
(2) definite value unit
Have qualification and in advance confirm the identical testing laboratory of its definite value ability through comparison by 6～8 families, adopt the Same Way demarcation that cooperates.
(3) definite value requirement
1) tackles each tested standard model and do independently mensuration respectively six to 8 times, measure and divide two unit, each unit to do independent mensuration three times; Be separated by between two unit and be no less than 3 days.6 data that obtain should be checked exceptional value by statistical method, as finding exceptional value are arranged, and should indicate, and do for supplement once and measure.Quote whole results then.
2) measurement instrument/surveying instrument of measuring of the definite value that is useful on need through calibrating or calibrate qualified, and before the deadline.
(4) data are handled
1) gathers whole raw data, investigate the normality that whole measurement data distribute.
2) under the situation that data Normal Distribution or similar normal state distribute, the mean value of the data of surveying in each laboratory is considered as the single measurement value, constitute one group of new measurement data.Reject dubious value with statistical method.Calculate population mean and standard deviation.
3) under total data Normal Distribution or similar normal state distribution situation, also be considered as one group of new measurement data.Reject dubious value, calculate population mean and the standard deviation of whole raw data again.
4) the definite value result of the antigen valence calibration result that cooperates sees Table 5.Gather whole raw data, investigate the normality that whole measurement data distribute.Through the descriptive statistics analysis, this batch antigen hemagglutinative titer is 300 ± 60, and final definite value is 320.
Table 5 cooperation calibration result
Annotate: numerical value is the highly diluted multiple of the whole aggegations of antigen in the table.
2. homogeneity check
Randomly draw 15 in sample, with its nt wt net weight of electronic analytical balance difference weighing, the results are shown in Table 6.Descriptive biometrics analysis draws: all between 0.102～0.12g, average is 0.114g to example weight; CV is 4.3%, all in 95% range of normal value; The result shows this batch standard items loading amount homogeneous.
Table 6 sample weighing analysis result
3. stability test
The results are shown in Table 7,8,9, the result shows that this antigen preserves 28 days HA valencys and do not descend under 37 ℃ of conditions, slightly reduces in 35 days.The HA valency did not descend when 4 ℃ of preservations were preserved 12 months in 6 months and-20 ℃.
Table 7 stability test result (one)
Table 8 stability test result (two)
Table 9 stability test result (three)
Appendix
Bird flu blood clotting (HA) and blood clotting suppress (HI) test
1 material
1.196 hole V-type (90 degree) micro-reaction plate, single track and multichannel micropipettor (being furnished with suction nozzle), loading slot, suction pipe, beaker etc.
1.20.85% physiological saline
1.2.1 8.5g NaCl is measured in the physiological saline preparation, adding distil water is to 1000ml;
1.2.2 it is with 121 ℃ of sterilization 30min, standby;
1.2.3 physiological saline once use, was no more than for 1 week in 2～8 ℃ of preservations.
1.3 A Shi (Alsevers) liquid claims glucose 2.05g, sodium citrate 0.8g, citric acid 0.055g, sodium chloride 0.42g, adding distil water is to 100ml, adjust pH to 6.1 after the heating for dissolving, and 69Kpa 15min autoclaving, 2～8 ℃ of preservations are standby.
2～3 SPF cocks of 1.41% chicken erythrocyte suspension collection or do not have bird flu and the blood of the healthy cock of antibody such as ewcastle disease mixes with equal-volume A Shi liquid, with 0.85% physiological saline washing 3 times, at every turn all with the centrifugal 5min of 3000rpm/min, the washing back is made into 1% (V/V) red cell suspension with physiological saline, and 2～8 ℃ of preservations are standby.
1.5 the antigen of antigen dissolving freeze-drying and serum all by the amount of specification mark on the label, are used physiological saline solution.
2 operation art formulas
2.1 blood clotting (HA) test
2.1.1 in the V-type micro-reaction plate, every hole adds 0.025ml physiological saline.
2.1.2 the 1st hole adds 0.025ml antigen, lashes mixing repeatedly 3～5 times.
Add the 2nd hole 2.1.3 draw 0.025ml antigen from the 1st hole, draw 0.025ml behind the mixing and add the 3rd hole, so carry out two-fold dilution to the 11 holes, 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 1～2min or gently detains the reaction plate mixed reactant at oscillator, under room temperature (20～25 ℃), leave standstill 20～30min or 2～8 ℃ of 45～60min.Result of determination when the control wells red blood cell significantly is button-type.
2.1.7 the result judges reaction plate 60 degree that tilt, and observes red blood cell and has or not the teardrop shape trickling, the highly diluted multiple that does not have teardrop sample trickling (100% aggegation) fully is hemagglutinative titer.
2.2 blood clotting suppresses (HI) test
2.2.1 according to tiring of HA test determination, calculate preparation 4 HAUs (4HAU) antigen.HA tires and is the extension rate that contains 4HAU antigen divided by 4.For example, it is 1: 256 that HA tires, and then the extension rate of 4HAU antigen should be 1: 64 (256 divided by 4).
2.2.2 the 1st～11 hole adds 0.025ml physiological saline, the 12nd hole adds 0.05ml physiological saline.
2.2.3 the 1st hole adds 0.025ml serum, fully inhales 0.025ml behind the mixing in the 2nd hole, two-fold dilution to the 10 holes are drawn 0.025ml from the 10th hole and are discarded successively.
2.2.4 the 1st～11 hole all adds the 4HAU antigen of 0.025ml, leaves standstill 30min or 2～8 ℃ of 50min under room temperature (20～25 ℃).
2.2.5 every hole adds the chicken erythrocyte suspension of 0.025ml 1% (V/V), the concussion mixing leaves standstill 20～30min or 2～8 ℃ of 45～60min under room temperature (20～25 ℃), and the contrast red blood cell will be obvious button-type and be sunken at the bottom of the hole.
3 results judgement is tired with the HI that the highest serum extension rate that suppresses 4HAU antigen fully is judged to this serum.Be no more than 1 titre when the HI of positive control serum tires with the known error of tiring, negative control sera is tired when not being higher than 2log2, and test can be set up.Tested serum HI tires≤and 3log2 is judged to feminine gender; That=4log2 is judged to is 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.
Claims (2)
1. bird flue virus H 5 N 1 subtype Re-5 strain virus hemagglutination-inhibition test antigen standard substance is characterized in that:
(1) this antigen standard substance contains the bird flue virus H 5 N 1 subtype Re-5 strain virus of deactivation;
(2) this antigen standard substance meets following standard: the antigen standard substance to the chicken red blood cell agglutination titer all 〉=8log2; This antigen can only be suppressed by avian influenza virus H5 hypotype Re-5 strain positive serum, and to avian influenza virus H9 hypotype, H7 hypotype positive serum, the reaction that all is negative of newcastle disease positive serum and egg drop syndrome virus-positive serum.
2. the preparation method of a bird flue virus H 5 N 1 subtype Re-5 strain virus hemagglutination-inhibition test antigen standard substance is characterized in that it being by utilizing by the bird flue virus H 5 N 1 subtype Re-5 strain virus of reorganization through following steps:
(1) preparation and the check of H5N1 hypotype Re-5 strain avian influenza virus seed culture of viruses; According to conventional method, the virus stain of making bird flue virus H 5 N 1 subtype Re-5 strain blood clotting inhibition antigen standard substance is reorganization bird flue virus H 5 N 1 subtype Re-5 strain, and seed culture of viruses copies with SPF chicken embryo; Seed culture of viruses should meet following standard through check:
1) HA tires: Re-5 strain seed culture of viruses is answered 〉=9log2 1% chicken red blood cell agglutination titer;
2) the every ml viral level of chicken embryo median infective dose answers 〉=10
7.0EID
50
3) identified for genes: AIVRe-5 strain HA gene cracking site amino acid sequence should be-RETR ↓-, meet typical low pathogenicity feature;
4) to the virulence of chicken embryo: Re-5 strain seed culture of viruses is made 10 times of serial dilutions to 10 with sterile saline
-4, with 10 piece of 10 age in days SPF chick embryo allantoic cavity of this concentration virus liquid inoculation, every embryo 0.1ml puts 36 ℃ and hatches 72h, and is should at least 9 pieces of chicken embryos not dead, and idiosome does not have the visible pathology of naked eyes;
5) to the virulence of chick: Re-5 strain seed culture of viruses is done 10 times of dilutions with sterile saline, 10 of intravenous inoculation SPF chickens in 5～6 age in week, every 0.1ml, intravenous inoculation pathogenic index are 0; 10 of collunarium inoculation SPF chickens in 3～4 age in week, every 0.1ml(contains 10
6EID
50), other gets 10 of the identical SPF chickens of condition, does not inoculate in contrast, with raising respectively under the condition, observes 14, death or obviously abnormal response all should not occur;
6) specificity: Re-5 strain seed culture of viruses should be positive reaction to avian influenza virus H5 hypotype antiserum, and avian influenza virus H1～H4, H6～H15 hypotype, newcastle disease virus and egg drop syndrome virus monospecific antiserum all should negatively be reacted;
7) pure property should not have bacterium, mould, mycoplasma and exogenous virus pollution;
(2) preparation of standard substance antigen and check;
1) preparation of antigen standard substance
According to conventional method, seed culture of viruses is inoculated SPF chicken embryo, results chicken blastochyle prepares bird flue virus H 5 N 1 subtype Re-5 strain blood clotting and suppresses the antigen semi-manufacture, through β-third Inner ester deactivation, concentrated, after carrying out steriling test, titration, by every bottle of 1ml packing freeze-drying, protective agent is for water for injection preparation sucrose concentration being 100% freeze drying protectant, after the filtration sterilization, protective agent and chicken blastochyle are prepared in the 1:9 ratio;
2) antigen check
1. physical behavior: antigen is the spongy loose agglomerate of white or canescence, easily breaks away from the bottle wall, adds dissolving rapidly behind the dilution;
2. steriling test: pick test, should there be bacterium or fungus growth;
3. titration: different parts is randomly drawed 3 samples and is carried out HA and measure during by freeze-drying, and each sample repeats to survey 2 times, and agglutination titer should all be not less than 8log2;
4. specificity check:; This antigen carries out hemagglutination-inhibition test to avian influenza virus H5 hypotype, H7 hypotype and H9 hypotype positive serum, newcastle disease positive serum, egg drop syndrome virus-positive serum respectively, this antigen can only be suppressed by bird flue virus H 5 N 1 subtype (Re-5 strain) positive serum, and to the reaction that all is negative of other positive serum;
5. residual moisture is measured: undertaken by " Chinese veterinary drug allusion quotation " residual moisture determination method, should be lower than 3%;
6. vacuum tightness is measured: undertaken by " Chinese veterinary drug allusion quotation " method, detect with high-frequency electronic spark instrument, and should be up to specification;
(3) definite value of standard substance
1) homogeneity check: freeze-drying quantity is 25 of 500 samplings when above; Freeze-drying quantity is 15 of 500 following time samplings; The nt wt net weight of every content of weighing is analyzed its homogeneity with statistical method respectively;
2) stability test: antigen is preserved under-20 ℃, 4 ℃, 37 ℃ conditions, and regularly carry out the HA valency and measure, each time all will be carried 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.
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