CN105603126A - Inactivation test method for inactivated avian influenza vaccine - Google Patents

Inactivation test method for inactivated avian influenza vaccine Download PDF

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CN105603126A
CN105603126A CN201610155427.8A CN201610155427A CN105603126A CN 105603126 A CN105603126 A CN 105603126A CN 201610155427 A CN201610155427 A CN 201610155427A CN 105603126 A CN105603126 A CN 105603126A
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cell
bottle
nutrient solution
avian influenza
cell bottle
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王玉玲
黄金妹
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FUZHOU DA BEI NONG BIOTECH Co Ltd
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Abstract

The invention discloses an inactivation test method for an inactivated avian influenza vaccine. The inactivation test method for the inactivated avian influenza vaccine comprises the following steps: adsorbing an inactivated avian influenza fluid on MDCK (Madin-Darby canine kidney) cells for 30-35 minutes; after adsorption, adding a DMEM (Dulbecco modified Eagle medium) culture solution into cell bottles, placing the cell bottles in a CO2 incubator at the temperature of 35 DEG C to be cultured for several days, harvesting a cell culture fluid, and freezing and thawing for three times; then carrying out continuous blind passage for three generations, and if cells in each cell bottle in the three generations have no cytopathic effect, an avian influenza antigen fluid is judged to be completely inactivated; and if any one cell bottle in the three generations has the cytopathic effect, determining that the avian influenza antigen fluid is judged to be incompletely inactivated. The inactivation test method for the inactivated avian influenza vaccine adopts an MDCK passage cell line for carrying out inactivation test on an inactivated avian influenza antigen, passage cells are stable and uniform, and MDCK cells not polluted by exogenous microorganisms are adopted, so that accuracy of a test result is guaranteed.

Description

A kind of deactivation method of inspection of inactivated avian influenza vaccine
Technical field
The invention belongs to medicine bioengineering for animals field, be specifically related to a kind of deactivation method of inspection of inactivated avian influenza vaccine.
Background technology
In recent years, country's great attention to safety control of bird flu to poultry bird flu compulsory immunization and aquaculture related personnel, China's bird flu epidemic situation is downward trend year by year generally. But at present, no matter be highly pathogenic bird flu or low pathogenicity bird flu, remain the formidable enemy of serious threat aviculture. Since two thousand four, inland of China is broken out highly pathogenic bird flu in succession, has not only given a heavy blow to domestic aviculture, and economy and Trade Development on China also caused serious impact.
Since 1933 utilize chicken embryo culture influenza virus to succeed, chicken embryo just becomes people and obtains in a large number the main source of influenza virus. But with chicken embryo separated flow Influenza Virus or go down to posterity and easily cause virus variation, and chicken embryo residue also may cause allergic reaction. Chicken embryo, as production of vaccine matrix, is supplied with difficulty and potential exogenous virus pollution problem while also there is large-scale production.
In recent years, along with frequently the breaking out and propagate across kind of bird flu, people recognize that need to study one can substitute chicken embryo when popular influenza is extensive, produces the cell culture system of influenza virus fast, in a large number. Application mdck cell system cultivates influenza virus and has not only solved the problem of chicken embryo protein legacy and exogenous virus pollution, and the virus immunity originality of cultivating is more stable.
Deactivation in veterinary biological product production, refers to BA, the fertility and pathogenic of destroy microorganisms, but does not affect as far as possible its immunogenicity, the microorganism being inactivated is mainly for the production of inactivated vaccine; Or refer to destroy the complement activity in diagnostic serum or serum to be checked, to avoid the interference effect of complement to diagnostic test.
Inactivator has specificity, some inactivator only has obvious deactivation to a part of microorganism, different inactivators is also different to the inactivating efficacy of same microorganism, as phenols can suppress and kill the brood body of most of bacterium, 5% carbolic acid solution can kill the gemma of bacterium within a few hours; Fungi and virus are not too responsive to phenols.
Therefore in avian influenza virus inactivated vaccine production process, just need to carry out deactivation inspection to the deactivation of antigen, to determine antigen, whether deactivation is complete, thereby avoids the incomplete vaccine of deactivation to cause bio-safety hidden danger.
Summary of the invention
The deactivation method of inspection that the object of the present invention is to provide a kind of inactivated avian influenza vaccine, the method can be stablized, and checks exactly the deactivation situation of bird flu antigen, is applied to the production testing of avian influenza virus inactivated vaccine.
For achieving the above object, the present invention adopts following technical scheme:
A deactivation method of inspection for inactivated avian influenza vaccine, comprises the following steps:
1) be taken to few 3 bottles of cell bottles and cultivate mdck cell 24-30h, mdck cell stand density reaches 80% when above, discards growth-promoting media, the phosphate buffer wash cell bottle of the pH7.2 of use 0.01mol/L 3-4 time;
2) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution of pancreatin, room temperature is placed 15-20min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 25-50 μ g/ml;
3) the bird flu antigen liquid after deactivation is done after 10 times of dilutions, respectively get 1-1.5mL and be inoculated in respectively in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30-35min, the bird flu antigen liquid after deactivation is adsorbed on mdck cell, jolting during this time 1-2 time, described CO2CO in incubator2Concentration be 5%;
4) after absorption, in each cell bottle, add respectively 8-10mlDMEM nutrient solution, cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 1st generation;
5) be taken to few 3 bottles of 24-30h that grown, and stand density reaches more than 80% mdck cell, discard growth-promoting media, with the phosphate buffer wash cell bottle of the pH7.2 of 0.01mol/L 3-4 time;
6) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution of pancreatin, room temperature is placed 15-20min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 25-50 μ g/ml;
7) get above-mentioned steps 4) cell culture fluid 1-1.5mL after freeze thawing, be inoculated in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30-35min, jolting during this time 1-2 time, described CO2CO in incubator2Concentration be 5%;
8) in each cell bottle, add respectively 8-10mlDMEM nutrient solution, cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 2nd generation;
9) repeat above-mentioned steps 5)-step 8), this is the 3rd generation of blind passage;
10) the deactivation method of inspection:
Result is judged: the each bottle of cell in 3 generations of blind passage in above-mentioned steps (cell of inoculation bird flu antigen liquid or cell culture fluid) all acellular pathology occurs, is judged to be the deactivation of bird flu antigen liquid complete; Respectively there is cytopathy for any 1 bottle of cell bottle, be judged to be the deactivation of bird flu antigen liquid incomplete.
Described cell bottle is T25 cell bottle.
In each cell bottle, the final concentration of described pancreatin in each cell bottle nutrient solution is 2.5-5.0 μ g/ml.
Described DMEM nutrient solution is that DMEM culture medium ultra-pure water is formulated, and regulates pH to 7.4. Described DMEM culture medium is that CIBCO company produces.
The present invention adopts above technical scheme, in prior art, the deactivation method of inspection of inactivated avian influenza vaccine adopts the inoculation of inoculation chicken allantoic cavity more, after cultivating certain hour, observe chicken embryo death situation and survey blood clotting situation, the method is because having related to chicken embryo, the not good meeting of chicken idioplasm amount causes non-specific death, and chicken embryo exists inoculating microbe also can cause the appearance of death or blood clotting phenomenon, all can make assay unstable or inaccurate. And the present invention adopts MDCK continuous cell line to carry out the deactivation inspection of avian influenza inactivation antigen, because of the stable and homogeneous of passage cell, assay is more stable; Another because adopting the mdck cell polluting without inoculating microbe, thus the accuracy of assay ensured.
Detailed description of the invention
A deactivation method of inspection for inactivated avian influenza vaccine, comprises the following steps:
1) be taken to few 3 bottles of cell bottles and cultivate mdck cell 24-30h, mdck cell stand density reaches 80% when above, discards growth-promoting media, the phosphate buffer wash cell bottle of the pH7.2 of use 0.01mol/L 3-4 time;
2) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution of pancreatin, room temperature is placed 15-20min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 25-50 μ g/ml;
3) the bird flu antigen liquid after deactivation is done after 10 times of dilutions, respectively get 1-1.5mL and be inoculated in respectively in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30-35min, the bird flu antigen liquid after deactivation is adsorbed on mdck cell, jolting during this time 1-2 time, described CO2CO in incubator2Concentration be 5%;
4) after absorption, in each cell bottle, add respectively 8-10mlDMEM nutrient solution, cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 1st generation;
5) be taken to few 3 bottles of 24-30h that grown, and stand density reaches more than 80% mdck cell, discard growth-promoting media, with the phosphate buffer wash cell bottle of the pH7.2 of 0.01mol/L 3-4 time;
6) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution of pancreatin, room temperature is placed 15-20min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 25-50 μ g/ml;
7) get above-mentioned steps 4) cell culture fluid 1-1.5mL after freeze thawing, be inoculated in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30-35min, jolting during this time 1-2 time, described CO2CO in incubator2Concentration be 5%;
8) in each cell bottle, add respectively 8-10mlDMEM nutrient solution, cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 2nd generation;
9) repeat above-mentioned steps 5)-step 8), this is the 3rd generation of blind passage;
10) the deactivation method of inspection:
Result is judged: the each bottle of cell in 3 generations of blind passage in above-mentioned steps (cell of inoculation bird flu antigen liquid or cell culture fluid) all acellular pathology occurs, is judged to be the deactivation of bird flu antigen liquid complete; Respectively there is cytopathy for any 1 bottle of cell bottle, be judged to be the deactivation of bird flu antigen liquid incomplete.
Described DMEM nutrient solution is that DMEM culture medium ultra-pure water is formulated, and regulates pH to 7.4.
In each cell bottle, the final concentration of described pancreatin in each cell bottle nutrient solution is 2.5-5.0 μ g/ml.
Embodiment 1
A deactivation method of inspection for inactivated avian influenza vaccine, comprises the following steps:
1) get 3 bottles of T25 cell bottles and cultivate mdck cell 24h, mdck cell stand density reaches 80% when above, discards growth-promoting media, the phosphate buffer wash cell bottle of the pH7.2 of use 0.01mol/L 3 times;
2) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution (pH7.4) of pancreatin, room temperature is placed 15min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 25 μ g/ml;
3) the bird flu antigen liquid after deactivation is done after 10 times of dilutions, respectively get 1mL and be inoculated in respectively in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30min, the bird flu antigen liquid after deactivation is adsorbed on mdck cell, jolting during this time 2 times, described CO2CO in incubator2Concentration be 5%;
4) after absorption, in each cell bottle, add respectively 10mlDMEM nutrient solution (pH7.4), cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 1st generation;
5) get 3 bottles of 24h that grown, and stand density reaches more than 80% mdck cell, discard growth-promoting media, with the phosphate buffer wash cell bottle of the pH7.2 of 0.01mol/L 3 times;
6) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution (pH7.4) of pancreatin, room temperature is placed 15min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 25 μ g/ml;
7) get above-mentioned steps 4) cell culture fluid 1mL after freeze thawing, be inoculated in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30min, jolting during this time 2 times, described CO2CO in incubator2Concentration be 5%;
8) in each cell bottle, add respectively 10mlDMEM nutrient solution (pH7.4), cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 2nd generation;
9) repeat above-mentioned steps 5)-step 8), this is the 3rd generation of blind passage;
10) the deactivation method of inspection:
Result is judged: the each bottle of cell in 3 generations of blind passage in above-mentioned steps (cell of inoculation bird flu antigen liquid or cell culture fluid) all acellular pathology occurs, is judged to be the deactivation of bird flu antigen liquid complete; Respectively there is cytopathy for any 1 bottle of cell bottle, be judged to be the deactivation of bird flu antigen liquid incomplete.
Experimental result: in 9 bottles of cell bottles in 3 generations of above-mentioned blind passage, every bottle of all acellular pathology appearance of cell, therefore can be judged to be the deactivation of bird flu antigen liquid complete.
Embodiment 2
A deactivation method of inspection for inactivated avian influenza vaccine, comprises the following steps:
1) get 4 bottles of T25 cell bottles and cultivate mdck cell 30h, mdck cell stand density reaches 80% when above, discards growth-promoting media, the phosphate buffer wash cell bottle of the pH7.2 of use 0.01mol/L 4 times;
2) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution (pH7.4) of pancreatin, room temperature is placed 20min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 50 μ g/ml;
3) the bird flu antigen liquid after deactivation is done after 10 times of dilutions, respectively get 1.5mL and be inoculated in respectively in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 35min, the bird flu antigen liquid after deactivation is adsorbed on mdck cell, jolting during this time 1 time, described CO2CO in incubator2Concentration be 5%;
4) after absorption, in each cell bottle, add respectively 8mlDMEM nutrient solution (pH7.4), cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 1st generation;
5) get 4 bottles of 30h that grown, and stand density reaches more than 80% mdck cell, discard growth-promoting media, with the phosphate buffer wash cell bottle of the pH7.2 of 0.01mol/L 4 times;
6) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution (pH7.4) of pancreatin, room temperature is placed 20min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 50 μ g/ml;
7) get above-mentioned steps 4) cell culture fluid 1.5mL after freeze thawing, be inoculated in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 35min, jolting during this time 1 time, described CO2CO in incubator2Concentration be 5%;
8) in each cell bottle, add respectively 8-10mlDMEM nutrient solution (pH7.4), cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 2nd generation;
9) repeat above-mentioned steps 5)-step 8), this is the 3rd generation of blind passage;
10) the deactivation method of inspection:
Result is judged: the each bottle of cell in 3 generations of blind passage in above-mentioned steps (cell of inoculation bird flu antigen liquid or cell culture fluid) all acellular pathology occurs, is judged to be the deactivation of bird flu antigen liquid complete; Respectively there is cytopathy for any 1 bottle of cell bottle, be judged to be the deactivation of bird flu antigen liquid incomplete.
Experimental result: in 12 bottles of cell bottles in 3 generations of above-mentioned blind passage, every bottle of all acellular pathology appearance of cell, therefore can be judged to be the deactivation of bird flu antigen liquid complete.
Embodiment 3
A deactivation method of inspection for inactivated avian influenza vaccine, comprises the following steps:
1) get 3 bottles of T25 cell bottles and cultivate mdck cell 28h, mdck cell stand density reaches 80% when above, discards growth-promoting media, the phosphate buffer wash cell bottle of the pH7.2 of use 0.01mol/L 3-time;
2) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution (pH7.4) of pancreatin, room temperature is placed 15min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 35 μ g/ml;
3) the bird flu antigen liquid after deactivation is done after 10 times of dilutions, respectively get 1mL and be inoculated in respectively in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30min, the bird flu antigen liquid after deactivation is adsorbed on mdck cell, jolting during this time 2 times, described CO2CO in incubator2Concentration be 5%;
4) after absorption, in each cell bottle, add respectively 8mlDMEM nutrient solution (pH7.4), cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 1st generation;
5) get 3 bottles of 28h that grown, and stand density reaches more than 80% mdck cell, discard growth-promoting media, with the phosphate buffer wash cell bottle of the pH7.2 of 0.01mol/L 3 times;
6) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution (pH7.4) of pancreatin, room temperature is placed 15min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 35 μ g/ml;
7) get above-mentioned steps 4) cell culture fluid 1mL after freeze thawing, be inoculated in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30min, jolting during this time 2 times, described CO2CO in incubator2Concentration be 5%;
8) in each cell bottle, add respectively 8-10mlDMEM nutrient solution, cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 2nd generation;
9) repeat above-mentioned steps 5)-step 8), this is the 3rd generation of blind passage;
10) the deactivation method of inspection:
Result is judged: the each bottle of cell in 3 generations of blind passage in above-mentioned steps (cell of inoculation bird flu antigen liquid or cell culture fluid) all acellular pathology occurs, is judged to be the deactivation of bird flu antigen liquid complete; Respectively there is cytopathy for any 1 bottle of cell bottle, be judged to be the deactivation of bird flu antigen liquid incomplete.
Experimental result: in 9 bottles of cell bottles in 3 generations of above-mentioned blind passage, cytopathy appears in the cell in wherein one bottle of cell bottle in the 3rd generation of blind passage, is therefore judged to be the deactivation of bird flu antigen liquid incomplete.

Claims (4)

1. a deactivation method of inspection for inactivated avian influenza vaccine, is characterized in that: it comprises the following steps:
1) be taken to few 3 bottles of cell bottles and cultivate mdck cell 24-30h, mdck cell stand density reaches 80% when above, discards growth-promoting media, the phosphate buffer wash cell bottle of the pH7.2 of use 0.01mol/L 3-4 time;
2) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution of pancreatin, room temperature is placed 15-20min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 25-50 μ g/ml;
3) the bird flu antigen liquid after deactivation is done after 10 times of dilutions, respectively get 1-1.5mL and be inoculated in respectively in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30-35min, the bird flu antigen liquid after deactivation is adsorbed on mdck cell, jolting during this time 1-2 time, described CO2CO in incubator2Concentration be 5%;
4) after absorption, in each cell bottle, add respectively 8-10mlDMEM nutrient solution, cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 1st generation;
5) be taken to few 3 bottles of 24-30h that grown, and stand density reaches more than 80% mdck cell, discard growth-promoting media, with the phosphate buffer wash cell bottle of the pH7.2 of 0.01mol/L 3-4 time;
6) in each cell bottle, add respectively 1ml to contain the DMEM nutrient solution of pancreatin, room temperature is placed 15-20min, and in described DMEM nutrient solution, the mass concentration of pancreatin is 25-50 μ g/ml;
7) get above-mentioned steps 4) cell culture fluid 1-1.5mL after freeze thawing, be inoculated in each cell bottle, cell bottle is placed in to the CO of 35 DEG C2In incubator, hatch 30-35min, jolting during this time 1-2 time, described CO2CO in incubator2Concentration be 5%;
8) in each cell bottle, add respectively 8-10mlDMEM nutrient solution, cell bottle is continued to be placed in above-mentioned CO2In incubator, continue to cultivate after 5 days, harvesting nutrient solution, and multigelation 3 times, this is blind passage 2nd generation;
9) repeat above-mentioned steps 5)-step 8), this is the 3rd generation of blind passage;
10) the deactivation method of inspection:
Result is judged: all acellular pathology appearance of the each bottle of cell in 3 generations of blind passage in above-mentioned steps, are judged to be the deactivation of bird flu antigen liquid complete; Respectively there is cytopathy for any 1 bottle of cell bottle, be judged to be the deactivation of bird flu antigen liquid incomplete.
2. the deactivation method of inspection of a kind of inactivated avian influenza vaccine according to claim 1, is characterized in that: described cell bottle is T25 cell bottle.
3. the deactivation method of inspection of a kind of inactivated avian influenza vaccine according to claim 1, is characterized in that: the final concentration of described pancreatin in each cell bottle nutrient solution is 2.5-5.0 μ g/ml.
4. the deactivation method of inspection of a kind of inactivated avian influenza vaccine according to claim 1, is characterized in that: described DMEM nutrient solution is that DMEM culture medium ultra-pure water is formulated, and regulates pH to 7.4.
CN201610155427.8A 2016-03-18 2016-03-18 Inactivation test method for inactivated avian influenza vaccine Pending CN105603126A (en)

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Citations (4)

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CN102000327A (en) * 2010-12-01 2011-04-06 齐鲁动物保健品有限公司 Mink viral enteritis inactivated vaccine and preparation method thereof
CN103122392A (en) * 2012-11-12 2013-05-29 福州大北农生物技术有限公司 Inactivation inspection method for inactivated vaccine of porcine circovirus
CN103924005A (en) * 2013-01-16 2014-07-16 辽宁成大生物股份有限公司 Detection method for influenza virus inactivation test verification

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余钧池、陈柳均: "流感病毒在MDCK细胞中培养的条件优化", 《国际检验医学杂志》 *
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