CN102050876A - Positive-serum and negative-serum standard substance of avian influenza virus H5N1 subtype Re-5 strain and preparation method thereof - Google Patents
Positive-serum and negative-serum standard substance of avian influenza virus H5N1 subtype Re-5 strain and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a positive-serum and negative-serum standard substance of avian influenza virus H5N1 subtype Re-5 strain and a preparation method thereof. The preparation method comprises the following steps of: preparing a standard substance positive serum (strongly positive serum and weakly positive serum): intramuscularly injecting an avian influenza virus H5N1 subtype Re-5 strain oil-emulsion inactivated vaccine in a young or adult SPF (Specific Pathogen Free) chicken, and then collecting the serum of the SPF chicken and carrying out semi-finished product inspection, adding a proper stabilizing agent and then freeze-drying; preparing negative serum: taking the blood of the SPF chicken, separating serum, carrying out semi-finished product inspection, and freeze-drying; and carrying out a series of technical processes, such as finished-product inspection, uniformity inspection, stability inspection, calibration and value determination of the standard substance and the like to obtain the positive-serum and negative-serum standard substance. The standard substance is the fundamental guarantee for the accurate diagnosis of avian influenza virus H5N1 subtype, the immune monitoring of a H5N1 subtype Re-5 strain and the accurate evaluation on the vaccine immune effect of the H5N1 subtype Re-5 strain, thereby improving the prevention and control level of avian influenza. The standard substance is the basic guarantee for the diagnosis of avian influenza virus H5N1 subtype and the evaluation and the quality control of the inspection working of related products.
Description
Technical field
The present invention relates to positive serum and the negative serum reference material and the preparation method of a kind of bird flue virus H 5 N 1 subtype Re-5 strain, 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 reference material is set up later, after 1970, European Pharmacopoeia comes out, and Britain is except that quoting international reference materials, also use the European Pharmacopoeia reference material, a small amount of reference material 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 standard 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 reference materials, and 1739 kinds of secondary reference materials relate to fields such as iron and steel, geology, oil, nuclear matter, environment, food and Clinical Laboratory.The reference material research of veterinary biologics lags behind, and have only 192 kinds at present, and the veterinary biologics check is almost nil with reference material.
Whether accurately the veterinary biologics reference material is control and definite veterinary biologics quality product, calibration verification testing instruments and method standard of physical, is correctly to diagnose transmissible disease, accurately monitor the basic substance 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 AIV H5N1 hypotype Re-5 strain is developed at the Highly Pathogenic Avian Influenza Virus (HPAIV) of China's current popular.Inactivated avian influenza vaccine with this strain development is a 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.But China does not still have the report of avian influenza virus H5 hypotype hemagglutination-inhibition test with reference material and correlative study thereof at present, external International Animal Health tissue reference laboratory such as (OIE) does not have yet, and different times detects the consistence 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 bird flu reference material, the diagnostic reagent reference material that particularly the H5 hypotype is relevant with the H9 hypotype, therefore it is particularly urgent to develop bird flue virus H 5 N 1 subtype blood clotting inhibition antigen reference material, this reference material is that the H 5 N 1 avian influenza hypotype is accurately diagnosed, AIV H5N1 hypotype Re-5 strain immunologic surveillance 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 reference material and " reference material management method " and the primary standard material technical specifications (JJG 1006-94) of following State Metrological Bureau's issue enforcement on July 10th, 1987, but this technical specifications is the development that is applicable to chemical ingredients, physics-chem characteristic and engineering characteristic primary standard material, for the biological product standards material, particularly veterinary biologics reference material development does not still have technical specifications at present and can follow.Therefore the development of veterinary biologics reference material is a footless research, has filled up the blank in the research field.
Summary of the invention
This aspect relates to a kind of bird flue virus H 5 N 1 subtype Re-5 strain positive serum and negative serum reference material, the positive serum reference material has strong positive serum reference materials and weak positive serum reference material, these two kinds of positive serum reference materials only are positive with bird flue virus H 5 N 1 subtype Re-5 strain antigen, the HI valency of strong positive serum reference materials is 〉=8log2, the HI valency of weak positive serum reference material is＜8log2 and 〉=4log2; These two kinds of positive serums and H7 and H9 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen all should negatively react; The negative serum reference material is under identical conditions, carry out hemagglutination inhibition test (HI test) with bird flu, newcastle disease, egg drop syndrome virus antigen respectively, serum is negative reaction to bird flu H5, H7, H9 hypotype, newcastle disease, egg drop syndrome virus antigen.
Technical scheme of the present invention:
(1) bird flue virus H 5 N 1 subtype Re-5 strain positive serum and negative serum reference material material standed for:
1) oil emulsion inactivated vaccine for preparing with bird flue virus H 5 N 1 subtype Re-5 strain virus according to a conventional method.
2) positive serum reference material candidate system is used young or adult SPF chicken, through intramuscular injection bird flue virus H 5 N 1 subtype R5 strain oil emulsion inactivated vaccine, preparation strong positive serum material standed for, after 2 immunity, choose the immune chicken of HI antibody titer 〉=8log2, gather chicken serum respectively and carry out mixing after the inspection of semifinished product freeze-drying behind the suitable stablizer of adding; Preparation weak positive serum material standed for only 1 immunity, choose HI antibody titer＜8log2 and 〉=the immune chicken of 4log2, gather chicken serum respectively and carry out mixing after the inspection of semifinished product, add freeze-drying behind the suitable stablizer.
3) SPF chicken blood picks up from negative serum system, and separation of serum also carries out the inspection of semifinished product, freeze-drying.
(2) strong positive serum, weak positive serum and the negative serum reference material material standed for of above preparation become strong positive serum, weak positive serum and negative serum reference material through a series of technological process such as the demarcation of check, homogeneity check and stability test and reference material, definite value.
Elaborating of technical solution of the present invention
One, the preparation of bird flue virus H 5 N 1 subtype Re-5 strain positive serum, weak positive serum and negative serum reference material
The 1 immunity preparation of vaccine
(1) preparation of viral liquid and check suppress antigenic preparation method's propagative viruses liquid according to bird flue virus H 5 N 1 subtype Re-5 strain standard blood clotting, and viral liquid is tested:
1) every bottle of chicken blastochyle of steriling test is taken a sample respectively, by People's Republic of China's veterinary drug allusion quotation (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 press, 2006, the present invention is called for short " Chinese veterinary drug allusion quotation ") method of regulation tests, and no bacterium or mould or mycoplasma are grown does 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.
(2) deactivation of viral liquid and check
1) deactivation filters the above-mentioned blastochyle that is up to the standards with sterilization multilayer gauze, be mixed in the large 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.
2) 5 of 10～11 age in days SPF chicken embryos are got in the deactivation check, inoculation deactivation blastochyle 0.1ml in each allantoic cavity, hatch 72h for 37 ℃, results chicken blastochyle is measured blood clotting and (is seen this specification sheets appendix for details, call " appendix " in the following text), negative, and 1 generation of blind passage, blood clotting measured, no blood clotting phenomenon, it is complete to be judged to deactivation.
(3) the import adjuvant that meets the biological products regulation is selected in the selection of adjuvant for use, and 80 and the sorbester p17 etc. that produce as moral home-made Marcol52 white oil, Singapore are emulsification adjuvant.
(4) protectant processing is pressed 6: 94 mixed post-heating to 130 ℃ 30min with sapn and white oil, and is standby after the aseptic cooling; Tween is standby behind autoclaving.
(5) tween after the preparation of vaccine will be sterilized earlier adds in the viral liquid that deactivation is up to the standards, with its mixing in 8% ratio; Quantitatively add the refrigerative white-oil adjuvant in the tissue mashing machine, low speed is opened tissue mashing machine, the viral liquid of the mixing tween amount by white-oil adjuvant 1/2 is slowly added in the tissue mashing machine, and the rotating speed with tissue mashing machine is increased to 8000r/min running 10min then.Be sub-packed in the vaccine bottle water-in-oil-type vaccine of preparation standby.
The preparation of 2 positive serums
(1) immunity selects for use 3～4 monthly age SPF fowl raisings in the negative pressure shield retaining, with 2 vaccinations of chest muscle injection system, dosage of inoculation is 1.0ml/ for the first time with the inactivated avian influenza vaccine for preparing, and carries out the inoculation second time at interval after 21 days, dosage is 1.5ml/, and the branch injection.
(2) examination blood is after the 2nd immunization 28 days, gathers serum with venous blood collection mode under the wing, and blood sampling is measured HI antibody titer (being undertaken by " appendix ") respectively simultaneously to chicken and serum reference numeral, selects HI antibody titer 〉=9log
2The SPF chicken standby.
(3) HI antibody titer 〉=9log is selected in the separation of serum
2The SPF chicken in the negative pressure shield retaining, carry out heart blood sampling with the 20ml asepsis injector, after finishing, blood collection places 37 ℃ of 4h earlier, and then place in 4 ℃ of refrigerators and spend the night, in the sterilisable chamber Bechtop, extract serum, if be mixed with red blood corpuscle in the serum, tackle it and carry out in the aseptic container that is mixed in after centrifugal after the same sterilization.
(4) the half-finished check of serum
1) steriling test is taken a sample to mixed serum, tests by " Chinese veterinary drug allusion quotation " prescriptive procedure, should not have bacterium or mould-growth.If living contaminants is arranged, the filter membrane of using 0.22 μ carries out filtration sterilization to it.
2) mensuration of HI antibody valence is carried out the mensuration of HI antibody valence by " Chinese veterinary drug allusion quotation " red cell agglutination inhibition method, and the result answers 〉=9log
2
3) specificity is identified serum to be checked is carried out hemagglutination inhibition test (HI test) (being undertaken by " appendix ") with H5, H7 and H9 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen respectively under identical conditions, only with the avian influenza virus H5 hypotype antigen (HI 〉=7log that is positive
2), all should negatively react with H7 and H9 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen (HI≤4log2).
4) serum of the preparation of serum through being up to the standards add trehalose (producing) and 0.5% by 3% of total amount by Beijing Baeyer enlightening biotech firm glycine (by Tianjin Kermel Chemical Reagent Co., Ltd.'s production) as lyophilized vaccine and stir; and test according to " Chinese veterinary drug allusion quotation " steriling test method, answer asepsis growth.
5) packing and freeze-drying are carried out packing with the antigen liquid for preparing with ampoule, and every bottle of 1ml puts into Freeze Drying Equipment rapidly and carries out vacuum freezedrying after the packing.Place-20 ℃ of preservations after pricking aluminium lid, label after the freeze-drying.
(5) inspection after construction
1) physical behavior is the spongy loose agglomerate of little yellow, easily separates with the bottle wall, adds dissolving rapidly behind the diluent, and the outward appearance after the dissolving should be identical with liquid serum.
2) steriling test is undertaken by " Chinese veterinary drug allusion quotation " steriling test method, answers asepsis growth.
3) titration is undertaken by appendix, and the HI antibody titer is answered 〉=8log2.
4) specificity is checked same work in-process, should be up to specification.
5) after PH mensuration is diluted serum by specification, measure its pH value, should be 7.9～8.2 with the pH instrument
6) residual moisture is measured by " Chinese veterinary drug allusion quotation " residual moisture assay method and is undertaken, and should be lower than 3%.
7) vacuum tightness is measured by " Chinese veterinary drug allusion quotation " vacuum tightness assay method and undertaken, and is up to specification.
8) definite value of serum HI antibody titer
1. valued methods hemagglutination inhibition test (HI 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 of cooperating.
3. definite value requirement
(a) tackle 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 obtained should be checked outlier by statistical method, as finding outlier are arranged, and should indicate, and do for supplement once and measure.Quote whole results then.
(b) 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 processing
(a) gather whole raw data, investigate the normality that whole take off data distribute.
(b) under the situation of data Normal Distribution or similar normal state distribution, each breadboard mean value of surveying data is considered as the single measurement value, constitutes one group of new take off data.Statistically reject dubious value with statistical method.Calculate population mean and standard deviation.
(c) under all data Normal Distribution or similar normal state distribution situation, also be considered as one group of new take off data.Statistically reject dubious value with statistical method, calculate the population mean and the standard deviation of whole raw data again.
9) 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.
10) stability test serum is preserved under-20 ℃, 4 ℃, 37 ℃ conditions respectively, and regularly carry out the HA valency and measure, each time all will be carried out the several replicate measurement, 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.
3. weak positive serum preparation
(1) immunity selects for use 3～4 monthly age SPF fowl raisings in the negative pressure shield retaining, and with the vaccination of chest muscle injection system, dosage of inoculation is 0.5ml/ with the inactivated avian influenza vaccine for preparing.
(2) examination blood is after immunization 14～21 days, gather serum with venous blood collection mode under the wing, blood sampling is measured HI antibody titer (being undertaken by " appendix ") respectively simultaneously to chicken and serum reference numeral, choose HI antibody titer＜8log2 and 〉=the immune chicken of 4log2 is standby.
(3) separation of serum choose HI antibody titer＜8log2 and 〉=the immune chicken of 4log2 carries out the heart blood sampling with the 20ml asepsis injector in the negative pressure shield retaining, after finishing, blood collection places 37 ℃ of 4h earlier, and then place in 4 ℃ of refrigerators and spend the night, in the sterilisable chamber Bechtop, extract serum, if be mixed with red blood corpuscle in the serum, tackle it and carry out in the aseptic container that is mixed in after centrifugal after the same sterilization.
(4) the half-finished check of serum
1) steriling test is taken a sample to mixed serum, tests by " Chinese veterinary drug allusion quotation ", should not have bacterium or mould-growth.If living contaminants is arranged, the filter membrane of using 0.22 μ carries out filtration sterilization to it.
2) mensuration of HI antibody valence is measured by appendix, and the result should be greater than 4log2, less than 8log2.
3) specificity is identified serum to be checked is carried out hemagglutination inhibition test (HI test) (being undertaken by " appendix ") with H5, H7 and H9 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen respectively under identical conditions, only be positive with avian influenza virus H9 hypotype antigen (HI antibody titer＜8log2 and 〉=4log2), all should negatively react with H7 and H5 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen.
(5) serum of the preparation of serum through being up to the standards add trehalose (producing) and 0.5% by 3% of total amount by Beijing Baeyer enlightening biotech firm glycine (by Tianjin Kermel Chemical Reagent Co., Ltd.'s production) as lyophilized vaccine and stir; and test according to " Chinese veterinary drug allusion quotation " steriling test method, answer asepsis growth.
(6) packing and freeze-drying are carried out packing with the antigen liquid for preparing with ampoule, and every bottle of 1ml puts into Freeze Drying Equipment rapidly and carries out vacuum freezedrying after the packing.Place-20 ℃ of preservations after pricking aluminium lid, label after the freeze-drying.
(7) inspection after construction
1) the same positive serum of physical behavior.
2) the same positive serum of steriling test.
3) titration is undertaken by appendix, HI antibody titer＜8log2 and 〉=4log2.
4) after pH mensuration is diluted serum by specification, measure its pH value, should be 7.6～8.2 with the pH instrument.
5) specificity is checked same positive serum.
6) residual moisture is measured same positive serum.
7) vacuum tightness is measured same positive serum.
8) the same positive serum of the definite value of serum HI antibody titer.
9) homogeneity is checked same positive serum.
10) the same positive serum of stability test.
4. negative serum
(1) serum separates young SPF fowl raising in the indoor negative pressure shield retaining of Biosafety experimentation on animals, in the indoor negative pressure shield retaining of Biosafety experimentation on animals, carry out the heart blood sampling with asepsis injector, after finishing, blood collection places 37 ℃ of 4h earlier, and then place in 4 ℃ of refrigerators and spend the night, in the sterilisable chamber Bechtop, extract serum, be mixed in then in the container after the same sterilization.
(2) the half-finished check of serum
1) steriling test is taken a sample to mixed serum, tests by " Chinese veterinary drug allusion quotation ", should not have bacterium or mould-growth.
2) titration is undertaken by appendix, and the HI antibody titer should be negative.
3) specificity (is carried out serum) under identical conditions by " appendix ", carry out hemagglutination inhibition test (HI test) with bird flu, newcastle disease, egg drop syndrome virus antigen respectively, serum is negative reaction to bird flu H5, H7, H9 hypotype, newcastle disease, egg drop syndrome virus antigen.
(3) preparation of serum add trehalose (producing) and 0.5% by 3% of total amount by Beijing Baeyer enlightening biotech firm glycine (by Tianjin Kermel Chemical Reagent Co., Ltd.'s production) as lyophilized vaccine and stir; and test by " Chinese veterinary drug allusion quotation " steriling test method, answer asepsis growth.
4) packing and freeze-drying are carried out quantitative packing by the 2ml/ bottle, rapidly vacuum freezedrying freeze-drying after the packing (by " People's Republic of China's veterinary biologics rules " (The Ministry of Agriculture of the People's Republic of China, MOA. in 2000 version of People's Republic of China's veterinary biologics rules. Chemical Industry Press, 2001, the present invention is called for short " rules ") carry out.
(5) inspection after construction
1) physical behavior is the spongy loose agglomerate of little yellow, easily separates with the bottle wall, adds dissolving rapidly behind the diluent, and the outward appearance after the dissolving should be identical with liquid serum.
2) steriling test is undertaken by " Chinese veterinary drug allusion quotation " steriling test method, answers asepsis growth.
3) the same positive serum of titration.
4) the same positive serum of specificity.
5) residual moisture is measured by " Chinese veterinary drug allusion quotation " residual moisture assay method and is undertaken, and should be lower than 3%.
6) vacuum tightness is measured by " Chinese veterinary drug allusion quotation " vacuum tightness assay method and undertaken, and is up to specification.
Two, the use of H9 hypotype positive serum, weak positive serum and negative serum reference material
Positive serum and weak positive serum system, gather the SPF chicken serum and carry out the inspection of semifinished product behind intramuscular injection bird flue virus H 5 N 1 subtype (R5 strain) oil emulsion inactivated vaccine with young SPF chicken, freeze-drying behind the suitable stablizer of adding; SPF chicken blood picks up from negative serum system, and separation of serum also carries out the inspection of semifinished product, and freeze-drying forms.
Be mainly used in: bird flue virus H 5 N 1 subtype Re-5 strain HI experimental control.
1. the dilution of standard 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 80,160,240,320,400,480,560 times of dilutions then successively.
2. hemagglutination test (HA test)
(1) get 96 hole V-type Microhemagglutination plates, each extent of dilution 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, there is the red corpuscle deposition all reacting holes bottom and is the teardrop shape trickling downwards along the scarp, presents the same person with the red corpuscle control wells and is not aggegation hole (-) fully; There is the red corpuscle deposition reacting hole bottom and slowly is teardrop shape trickling person downwards along the scarp is incomplete aggegation hole (+); There is the red corpuscle deposition reacting hole bottom but is not 50% aggegation hole (++) along scarp trickling person; Except that a little red corpuscle was arranged at the bottom, other positions of reacting hole were almost complete aggegation hole person (+++); The no red corpuscle 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) 4HA
100Antigenic preparation and checking
1) antigen diluent method
Antigen is carried out 10 times of dilutions earlier, and redilution is for containing 4HA
100Hemagglutinin.4HA with preparation
100Hemagglutinin 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 extent of dilution 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 1min～2min.
6) room temperature (24～26 ℃) is placed 30min, result of determination.
7) criterion: if the antigen liquid of preparation is 4HA
100, then 1: 4 extent of dilution will provide 100% aggegation point, i.e. the no red corpuscle in reacting hole bottom, the complete aggegation in entire reaction hole; If 4HA
100Be higher than 4 units, possibility was 100% an aggegation point in 1: 5 or 1: 6; If lower, then possibility was 100% an aggegation point in 1: 2 or 1: 3.Should suitably adjust according to measurement result, its accurate extension rate=make extension rate * 240/4 of 100% red cell agglutination terminal point makes the antigen working fluid really be 4HA
100
(2) dilution of serum
1) standard positive serum dilute serum recover commercial weight after, carry out 10 times, 20 times dilutions successively; Carry out 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) after the dilute serum of tested serum recovers commercial weight, carry out 10 times, 20 times dilutions successively; Carry out 160,240,320,400,480,560 times then ... increase progressively 80 times of dilutions successively.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.
(3) all serum holes add 4HA on the V-type micro-reaction plate
100Antigen liquid 25 μ L.Last 1 row establishes 50 μ L physiological saline and 25 μ L physiological saline+25 μ L 4HA
100Antigen (each 4 hole) is done contrast.
(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 corpuscle deposition all reacting holes bottom and is the teardrop shape trickling downwards along the scarp, presents the same person with the red corpuscle control wells and is not aggegation hole (-) fully; There is the red corpuscle deposition reacting hole bottom and slowly is teardrop shape trickling person downwards along the scarp is incomplete aggegation hole (+); There is the red corpuscle deposition reacting hole bottom but is not 50% aggegation hole (++) along scarp trickling person; Except that a little red corpuscle was arranged at the bottom, other positions of reacting hole were almost complete aggegation hole person (+++); The no red corpuscle in reacting hole bottom, the complete aggegation person in entire reaction hole is complete aggegation hole (#).
(6) conclusion
Positive serum is 200 ± 40 times, and the negative serum dilution holes is (+), (++), (+++) or (#); 50 μ L physiological saline control wells are (-), 25 μ L physiological saline+25 μ L 4HA
100The antigen hole is (#), and this moment, test can be judged to establishment.The red cell agglutination that the high dilution that is edited the hole of the not aggegation fully correspondence of 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 positive serum and negative serum reference material and preparation method.This reference material positive serum and weak positive serum system, gather the SPF chicken serum and carry out the inspection of semifinished product behind intramuscular injection bird flue virus H 5 N 1 subtype (Re-5 strain) oil emulsion inactivated vaccine with young or adult SPF chicken, freeze-drying behind the suitable stablizer of adding; SPF chicken blood picks up from negative serum system, and separation of serum also carries out the inspection of semifinished product, freeze-drying; Through a series of technological processs such as the demarcation of inspection after construction, homogeneity check and stability test and reference material, definite values and make.This reference material is that bird flu H5 hypotype is accurately diagnosed, the immunologic surveillance of H5N1 hypotype (Re-5 strain) reaches the basic guarantee that its immune effect of vaccine is correctly estimated, and improves the prevention and control level of bird flu.Diagnose inspection routine to estimate basic guarantee with quality control to bird flu H5 hypotype with related products.
Embodiment 1
Positive serum is made and the inspection of semifinished product
1. positive serum manufacturing
(1) immunity selects for use 3～4 monthly age SPF fowl raisings in the negative pressure shield retaining, with 2 vaccinations of chest muscle injection system, dosage of inoculation is 1.0ml/ for the first time with the inactivated avian influenza vaccine for preparing, and carries out the inoculation second time at interval after 21 days, dosage is 1.5ml/, and the branch injection.
(2) examination blood is after the 2nd immunization 28 days, gathers serum with venous blood collection mode under the wing, and blood sampling is measured HI antibody titer (being undertaken by " appendix ") respectively simultaneously to chicken and serum reference numeral, and the SPF chicken that selects HI antibody titer 〉=9log2 is standby.
(3) separation of serum selects the SPF chicken of HI antibody titer 〉=9log2 to carry out the heart blood sampling with the 20ml asepsis injector in the negative pressure shield retaining, after finishing, blood collection places 37 ℃ of 4h earlier, and then place in 4 ℃ of refrigerators and spend the night, in the sterilisable chamber Bechtop, extract serum, if be mixed with red blood corpuscle in the serum, tackle it and carry out in the aseptic container that is mixed in after centrifugal after the same sterilization.
2. the half-finished check of positive serum
(1) steriling test is taken a sample to mixed serum, tests by " Chinese veterinary drug allusion quotation ", should not have bacterium or mould-growth.
(2) mensuration of HI antibody valence is carried out the mensuration of HI antibody valence by appendix, the result should 〉=9log2.
(3) specificity is identified serum to be checked is carried out hemagglutination inhibition test (HI test) (being undertaken by " appendix ") with H5, H7 and H9 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen respectively under identical conditions, only be positive with avian influenza virus H9 hypotype antigen (HI 〉=7log2), all should negatively react with H7 and H9 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen (HI≤4log2).
(4) serum of the preparation of serum through being up to the standards add trehalose (producing) and 0.5% by 3% of total amount by Beijing Baeyer enlightening biotech firm glycine (by Tianjin Kermel Chemical Reagent Co., Ltd.'s production) as lyophilized vaccine and stir; and test by " Chinese veterinary drug allusion quotation " steriling test method, answer asepsis growth.
(5) packing and freeze-drying are carried out packing with the antigen liquid for preparing with ampoule, and every bottle of 1ml puts into Freeze Drying Equipment rapidly and carries out vacuum freezedrying after the packing.Place-20 ℃ of preservations after pricking aluminium lid, label after the freeze-drying.
Embodiment 2
Weak positive serum is made and the inspection of semifinished product
1. weak positive serum manufacturing
(1) immunity selects for use 3～4 monthly age SPF fowl raisings in the negative pressure shield retaining, and with the vaccination of chest muscle injection system, dosage of inoculation is 0.5ml/ with the inactivated avian influenza vaccine for preparing.
(2) examination blood is after immunization 14～21 days, gathers serum with venous blood collection mode under the wing, and blood sampling is measured HI antibody titer (being undertaken by appendix) respectively simultaneously to chicken and serum reference numeral, and the SPF chicken that selects the HI antibody titer and be 5log2 is standby.
(3) separation of serum selects the SPF chicken of HI antibody titer 5log2 to carry out the heart blood sampling with the 20ml asepsis injector in the negative pressure shield retaining, after finishing, blood collection places 37 ℃ of 4h earlier, and then place in 4 ℃ of refrigerators and spend the night, in the sterilisable chamber Bechtop, extract serum, if be mixed with red blood corpuscle in the serum, tackle it and carry out in the aseptic container that is mixed in after centrifugal after the same sterilization.
2. the half-finished check of weak positive serum
(1) steriling test is taken a sample to mixed serum, tests by " Chinese veterinary drug allusion quotation ", should not have bacterium or mould-growth.If living contaminants is arranged, the filter membrane of using 0.22 μ carries out filtration sterilization to it.
(2) mensuration of HI antibody valence is carried out the mensuration of HI antibody valence by appendix, and the result should be HI valency＜8log2, and＞4log2.
(3) specificity is identified serum to be checked is carried out hemagglutination inhibition test (HI test) (being undertaken by " appendix ") with H5, H7 and H9 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen respectively under identical conditions, only with the avian influenza virus H9 hypotype antigen (HI valency＜8log2 that is positive, and＞4log2), all should negatively react with H7 and H9 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen.
(4) serum of the preparation of serum through being up to the standards add trehalose (producing) and 0.5% by 3% of total amount by Beijing Baeyer enlightening biotech firm glycine (by Tianjin Kermel Chemical Reagent Co., Ltd.'s production) as lyophilized vaccine and stir; and test by " Chinese veterinary drug allusion quotation " steriling test method, answer asepsis growth.
(5) packing and freeze-drying are carried out packing with the antigen liquid for preparing with ampoule, and every bottle of 1ml puts into Freeze Drying Equipment rapidly and carries out vacuum freezedrying after the packing.Place-20 ℃ of preservations after pricking aluminium lid, label after the freeze-drying.
Embodiment 3
Negative serum is made and the inspection of semifinished product
1. negative serum manufacturing
Young SPF fowl raising is in the indoor negative pressure shield retaining of Biosafety experimentation on animals, in the indoor negative pressure shield retaining of Biosafety experimentation on animals, carry out the heart blood sampling with asepsis injector, after finishing, blood collection places 37 ℃ of 4h earlier, and then place in 4 ℃ of refrigerators and spend the night, in the sterilisable chamber Bechtop, extract serum, be mixed in then in the container after the same sterilization.
2. the half-finished check of negative serum
(1) the bacterium check is tested by " Chinese veterinary drug allusion quotation " to mixed serum sampling, should not have bacterium or mould-growth.
(2) titration is pressed appendix and is measured the HI antibody titer, should be negative.
(3) specificity (is carried out serum) under identical conditions by " appendix ", carry out hemagglutination inhibition test (HI test) with bird flu, newcastle disease, egg drop syndrome virus antigen respectively, serum is negative reaction to bird flu H5, H7, H9 hypotype, newcastle disease, egg drop syndrome virus antigen.
(4) preparation of serum add trehalose (producing) and 0.5% by 3% of total amount by Beijing Baeyer enlightening biotech firm glycine (by Tianjin Kermel Chemical Reagent Co., Ltd.'s production) as lyophilized vaccine and stir; and test by " Chinese veterinary drug allusion quotation " steriling test method, answer asepsis growth.
(5) packing and freeze-drying are carried out quantitative packing by the 2ml/ bottle, vacuum freezedrying freeze-drying (carrying out according to " rules " method) rapidly after the packing.
Embodiment 4
The serum inspection after construction
1. physical behavior assay
Be the spongy loose agglomerate of little yellow, easily break away from, add dissolving rapidly behind the diluent with the bottle wall.
2. steriling test result
Assay sees Table 1, and all freeze-dried products all are negative.
Table 1 steriling test cartogram
3. titration result
Measurement result sees Table 2,3,4.The result shows, H 5 N 1 avian influenza hypotype (Re-5 strain) positive serum is tired and is that 8log2, weak positive serum tire and is that it is 0 that 6log2, negative serum tire.
The table 2 01 positive serum detected result of tiring
Annotate: " # " represents whole aggegations in the table, all aggegations of " ++ " expression part, and "-" represents not aggegation.
The table 301 weak positive serum detected result of tiring
Annotate: " # " represents whole aggegations in the table, all aggegations of " ++ " expression part, and "-" represents not aggegation.
The table 4 01 negative serum detected result of tiring
Annotate: " # " represents whole aggegations in the table, all aggegations of " ++ " expression part, and "-" represents not aggegation.
4. specificity assay
The results are shown in Table 5, the result shows that positive serum and weak positive serum can only be suppressed by H5 hypotype antigen, and to the reaction that all is negative of other antigen; Negative serum is to the reaction that all is negative of all antigens.
Table 5 antigen-specific inspection statistics table
Annotate: " # " represents whole aggegations in the table, and promptly antigen and serum do not produce inhibited reaction; "-" represents not aggegation, and promptly antigen and serum produce inhibited reaction.
5. residual moisture measurement result
Assay sees Table 6, and the result shows, the content of the residue moisture content of all samples is all less than 3%, the requirement of conformance with standard material.
Table 6 residue moisture content assay
6. vacuum tightness measurement result
All peace bottle vacuum tightnesss all meet the requirements.
Embodiment 5
1. the definite value of tiring of reference material
(1) valued methods hemagglutination inhibition test (HI 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 of cooperating.
(3) definite value requirement
1) tackle each tested standard model and do independently mensuration respectively six to eight times, divide two unit, each unit is done independent mensuration three times; Be separated by between two unit and be no less than three days.Six data that obtained should be checked outlier by statistical method, as finding outlier 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 processing
1) gathers whole raw data, investigate the normality that whole take off data distribute.
2) under the situation of data Normal Distribution or similar normal state distribution, each breadboard mean value of surveying data is considered as the single measurement value, constitutes one group of new take off data.Statistically reject dubious value with statistical method.Calculate population mean and standard deviation.
3) under all data Normal Distribution or similar normal state distribution situation, also be considered as one group of new take off data.Statistically reject dubious value with statistical method, calculate the population mean and the standard deviation of whole raw data again.
(5) the definite value result of serum titer
The cooperation calibration result sees Table 7,8,9.Gather whole raw data, investigate the normality that whole take off data distribute.Through the descriptive statistics analysis, it is 200 ± 40 that this batch positive serum blood clotting suppresses to tire, and final definite value is 240; It is 50 ± 10 that this batch weak positive serum blood clotting suppresses to tire, and final definite value is 60; It is 0 that this batch negative serum blood clotting suppresses to tire.
Table 7 cooperation calibration result
Annotate: numerical value is the highly diluted multiples of the whole agglutinative of antigen in the table.
Table 8 cooperation calibration result
Annotate: numerical value is the highly diluted multiples of the whole agglutinative of antigen in the table.
Table 9 cooperation calibration result
2. homogeneity check
Randomly draw each 15 in positive serum, weak positive serum and negative serum sample,, the results are shown in Table 2-10 with its nt wt net weight of electronic analytical balance difference weighing.Descriptive biometrics analysis draws: all between 0.094～0.107g, mean number is 0.099g to the positive serum example weight, and CV is 4.0%, all in 95% range of normal value; All between 0.087～0.096g, mean number is 0.091g to the weak positive serum example weight, and CV is 3.3%, all in 95% range of normal value; All between 0.105～0.123g, mean number is 0.114g to the negative serum example weight, and CV is 4.4%, all in 95% range of normal value; The result shows positive serum, weak positive serum and negative serum standard substance loading amount homogeneous.
Table 10 sample weighing analytical results
3 stability tests
The results are shown in Table 11,12,13, the result shows that positive serum and weak positive serum preserve 2 months HI valencys and do not descend under 37 ℃ of conditions, slightly reduces in 3 months; The HI valency did not reduce when 4 ℃ of preservations were preserved 12 months in 6 months and-20 ℃.
Table 11 stability test result (one)
Table 12 stability test result (two)
Table 13 stability test result (three)
Appendix
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 8.5g NaCl is measured in the physiological saline preparation, adding distil water is to 1000ml;
1.2.2 it is, standby with 121 ℃ of sterilization 30min;
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, Trisodium Citrate 0.8g, citric acid 0.055g, sodium-chlor 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 newcastle 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 antigen dissolves freeze dried antigen and serum all by the amount of specification mark on the label, uses 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, 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 from the 1st hole.
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 Sptting plate is shaken 1～2min or gently detains the Sptting plate mixed reactant on vibrator, under room temperature (20～25 ℃), leave standstill 20～30min or 2～8 ℃ of 45～60min.Result of determination when the control wells red corpuscle significantly is button-type.
2.1.7 the result judges Sptting plate 60 degree that tilt, and observes red corpuscle and has or not the teardrop shape trickling, the highly diluted multiple that does not have teardrop sample trickling (100% aggegation) fully is a hemagglutinative titer.
2.2 blood clotting suppresses (HI) test
2.2.1, calculate preparation 4 HAUs (4HAU) antigen according to tiring of HA test determination.HA tires to be divided by 4 and contains the antigenic extension rate of 4HAU.For example, it is 1: 256 that HA tires, and then the antigenic extension rate of 4HAU 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 corpuscle will be obvious button-type and be sunken at the bottom of the hole.
3 results judge to suppress the HI that the antigenic highest serum extension rate of 4HAU is judged to this serum fully and tire.When the HI of positive control serum tires and the known error of tiring is no more than 1 titre, 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 (3)
1. bird flue virus H 5 N 1 subtype Re-5 strain positive serum and negative serum reference material is characterized in that:
(1) the positive serum reference material has strong positive serum and weak positive serum reference material, these two kinds of positive serum reference materials only are positive with bird flue virus H 5 N 1 subtype Re-5 strain antigen, HI valency 〉=the 8log2 of strong positive serum, the HI valency＜8log2 of weak positive serum and 〉=4log2; These two kinds of positive serums and H7 and H9 subtype avian influenza, newcastle disease, egg drop syndrome virus antigen all should negatively react;
(2) the negative serum reference material is under identical conditions, carry out hemagglutination inhibition test (HI test) with bird flu, newcastle disease, egg drop syndrome virus antigen respectively, serum is negative reaction to bird flu H5, H7 and H9 hypotype, newcastle disease, egg drop syndrome virus antigen.
2. the preparation method of bird flue virus H 5 N 1 subtype Re-5 strain positive serum and negative serum reference material is characterized in that:
(1) oil emulsion inactivated vaccine for preparing with bird flue virus H 5 N 1 subtype Re-5 strain virus according to a conventional method;
(2) positive serum reference material candidate system is with young or adult SPF chicken, through intramuscular injection bird flue virus H 5 N 1 subtype Re-5 strain oil emulsion inactivated vaccine:
1) preparation strong positive serum material standed for after 2 immunity, is chosen the immune chicken of HI antibody titer 〉=8log2, gathers chicken serum respectively and carries out mixing after the inspection of semifinished product freeze-drying behind the suitable stablizer of adding;
2) preparation weak positive serum material standed for only 1 immunity, choose HI antibody titer＜8log2 and 〉=the immune chicken of 4log2, gather chicken serum respectively and carry out mixing after the inspection of semifinished product, add freeze-drying behind the suitable stablizer;
(3) SPF chicken blood picks up from negative serum system, and separation of serum also carries out the inspection of semifinished product, freeze-drying;
(4) strong positive serum, weak positive serum and the negative serum reference material material standed for of above preparation become strong positive serum, weak positive serum and negative serum reference material through a series of technological process such as the demarcation of check, homogeneity check and stability test and reference material, definite value.
3. as the described a kind of bird flue virus H 5 N 1 subtype Re-5 strain positive serum of claim 1-2 and negative serum reference material and preparation method, it is characterized in that the lyophilized vaccine that uses is: press 3% of serum total amount and add the glycine of trehalose and 0.5% as lyophilized vaccine.
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