This strain is inoculated with primary hamster kidney monolayer cells with a rabies fixed virus (aG) adapted strain. The virus liquid is harvested after cultivation, inactivated by adding formaldehyde solution, concentrated, and then made of aluminum hydroxide. Used to prevent rabies.
1 poison
1.1 Source of poison
The rabies fixed virus aG adaptive strain was adapted from the rabies fixed virus Beijing strain after being passaged in hamster kidney cells, and then alternately adapted in the guinea pig brain. It is verified, preserved and distributed by the China National Institute for the Control of Pharmaceutical and Biological Products. Subsequent generations of production virus should be replaced by no more than 10aG.
1.2 Verification of poison
1.2.1 Sterility test
The aG virus must be tested for sterility after each passage and post-mortem is accepted.
1.2.2 Virus titration
The aG virus strain is titrated with a mouse with a body weight of 11 to 13 g, and the titer ≥8.0 LogLD50 / 1.0 mL can be used for production.
1.2.3 Pure poison test
Before and at the end of production, mice are used for in-brain neutralization tests to identify the specificity of the poisonous species. The specific antiserum used shall be provided by the China National Institute for the Control of Pharmaceutical and Biological Products. The neutralization index must be above 500. If there is any doubt about the poisonous species during the production process, it should be identified in time.
1.3 Passage of virus species
Choose healthy guinea pigs weighing 120-160g, inject in the brain, select those with typical rabies symptoms with an incubation period of 80-100 hours to bleed to death, and take the brain aseptically. The guinea pig brain is passed for 5 consecutive generations.
1.4 Preservation of virus
The aG strain for production use should be kept below -60 ℃ for no more than 1 year.
2 Vaccine manufacturing
2.1 Cell preparation
2.1.1 Gopher
Choose healthy hamsters from 12 to 14 days of age, such as hyperemia, exudate, and abnormal kidneys in the abdominal cavity should be discarded.
2.1.2 Anatomy
The hamsters were sacrificed, and the fur was washed several times with disinfectant. The final soak was performed for a certain period of time. The kidneys were taken aseptically and shredded in a bottle.
2.1.3 Cell digestion and packaging
Wash the chopped kidney block several times, add the appropriate amount of trypsin digestion solution, leave at 2-8 ° C overnight (about 16-20 hours) or 37 ° C water bath to digest for an appropriate time, discard the digestion solution, and wash it 2 to 3 times with the washing solution , Shake or pipette to disperse the cells, add the appropriate amount of growth solution, divide into flasks, and incubate at 37 ℃ ± 0.5 ℃ to grow into a single layer.
2.1.4 Using liquid
Neither should contain penicillin or other β-lactam antibiotics.
2.1.4.1 Growth fluid
In order to contain not more than 10% calf serum hydrolyzed milk protein SMI solution or other suitable culture solution, an appropriate amount of antibiotics may be added.
2.1.4.2 Soaking solution
In order to hydrolyze milk protein SMI solution or other suitable culture solution, the protective agent can be more than 0.1% human albumin, and an appropriate amount of antibiotics can be added.
2.1.4.3 Maintenance fluid
It is 199 or other suitable solution, in which more than 0.4% of human albumin can be used in the protective agent, and appropriate antibiotics can be added.
2.1.5 Exogenous factor inspection
Leave 5% (or not less than 500 mL) suspension per cell of the production batch as a control cell to check for foreign viruses. Except that the cells are not inoculated with virus, the cell concentration and treatment are carried out in parallel with the production process. Observe with a microscope until the day the stock solution is harvested, and no lesions should occur. And 0.2% ~ 0.5% guinea pig erythrocytes (stored at 4 ℃ not more than 7 days) for blood adsorption test, set at 4 ~ 8 ℃ for 30 minutes to determine the results; then set at 20 ~ 25 ℃ for 30 minutes to determine the results again, all should be negative. If there are suspicious lesions or suspicious positive blood adsorption, continue to pass blindly on the same kind of cells. If the virus is transmitted, the vaccine prepared by the batch of cells should be discarded.
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