New cattle serum test requirements for a large inventory
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This product is derived from the blood of newborn calves that have not been fed within 14 hours of birth. The serum is processed through sterilization and filtration, ensuring no additional substances are introduced during production. Before use in manufacturing, each batch of newborn bovine serum must undergo comprehensive testing to meet regulatory standards. The protein concentration should range between 3.5% and 5.0%. Hemoglobin content is measured using the cyanide high-iron method or an equivalent technique. Both plaque assays and proliferation assays must remain below 0.02%. The serum must be free from phage contamination and should not contain E. coli phage, with specific indicators determined as needed. Sterility checks must comply with legal requirements, and mycoplasma testing must also be conducted according to regulations. Additionally, bacterial endotoxin levels should not exceed 10 EU/ml, and virus screening is essential.
Virus Screening
Cell Culture Method
1. Non-blood-adsorbed Virus Test: Newborn bovine serum samples are inoculated into human, monkey, and bovine kidney cell lines. Bovine kidney cells must be confirmed free from bovine viral contamination. At least six bottles of each cell type are inoculated, with each culture receiving at least 25% of the total medium volume. Cultures are incubated at 36 ± 1°C for 21 days. A negative control and a virus-positive control are included for each cell line. During the process, media can be changed based on cell growth, and cytopathic effects (CPE) are observed before each change. After the incubation period, the negative control should show no CPE, while the positive control must display typical CPE. If the test sample shows no CPE, it meets the requirement.
2. Blood-adsorbed Virus Test: Two bottles of each cell type are inoculated with the test sample for this test. A mixture of 0.2%–0.5% chicken and guinea pig red blood cells is applied to the cell surface. The cells are first incubated at 2–8°C for 2 minutes, then moved to 20–25°C for 30 minutes. Microscopic examination is used to check for red blood cell adsorption, which should be negative. A blood-adsorption positive control is also set up simultaneously.
Immunofluorescence Antibody Test
1. Cell Growth Curve Determination: A 10% cell culture medium is prepared, and cells are seeded at a density of 10^4 per ml. Live cell counts are taken daily for one week, and a growth curve is plotted. The maximum cell concentration should reach at least 10^6 per ml.
2. Cell Doubling Time Calculation: Using the growth curve, the doubling time is calculated. The formula is: Doubling Time = T / log2(Y/X), where Y is the cell count at peak, X is the initial cell count, and T is the time before the peak. For SP2/0 cells, the doubling time should not exceed 20 hours.
3. Cloning Efficiency Determination: Cells are diluted using the limiting dilution method and seeded into 96-well plates at a density of 1 cell per well. At least 48 wells per plate are used, and cultures are maintained at 37°C with 5% CO₂ for one week. The cloning efficiency should be at least 70%, calculated as (A/B) × 100%, where A is the number of growing cells and B is the total number of cells inoculated.
This detailed testing ensures the safety and quality of the newborn bovine serum, making it suitable for various biotechnological and research applications.