New cattle serum test requirements for a large inventory
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This product is manufactured by collecting blood from newborn calves that have not been fed within 14 hours of birth. The serum is then sterilized and filtered to ensure purity. No additional substances may be added during the production process, ensuring the integrity of the final product. Before being used in production, each batch of newborn bovine serum must undergo comprehensive testing to meet regulatory standards. The protein content should range between 3.5% and 5.0%. Hemoglobin levels are determined using the cyanide high iron method or another suitable technique. Both plaque assays and proliferation assays must remain below 0.02%, and the serum must be free from phage contamination. Additionally, it should be tested for E. coli phage osmotic pressure (with an indicator to be determined). Sterility checks must follow legal requirements, and mycoplasma testing must also comply with established regulations. Bacterial endotoxin levels should not exceed 10 EU/ml, and virus testing must be conducted as per standard procedures.
**Virus Testing**
**1. Non-blood-adsorbed Virus Test**
The test samples are inoculated into human, monkey-derived, and bovine kidney cell lines. The bovine kidney cells must first be confirmed to be free of bovine viral contamination. At least six bottles of each cell type are inoculated, with each inoculum comprising at least 25% of the total culture volume. The cultures are incubated at 36 ± 1°C for 21 days. A negative control and a virus-positive control must be included for each cell line. During the culture period, the medium can be replaced based on cell growth, and cytopathic effects must be observed before each change. After the incubation period, the negative control should show no cytopathic changes, while the positive control should exhibit typical cytopathic effects. If the test sample shows no cytopathic effects, it is considered acceptable.
**2. Blood-adsorbed Virus Test**
Two bottles of each of the above-mentioned cell lines are inoculated with the test sample and subjected to a blood-adsorption virus test. A mixture of 0.2%–0.5% chicken and guinea pig red blood cells is placed on the surface of the cells. The cells are incubated at 2–8°C for 2 minutes, followed by incubation at 20–25°C for 30 minutes. Microscopic examination is performed to check for red blood cell adsorption, which should be negative. A blood-adsorption positive control must also be included.
**Immunofluorescence Antibody Test**
**1. Cell Growth Curve Determination**
A 10% cell culture medium is prepared, and cells are inoculated at a concentration of 10ⴠcells per ml. Living cells are counted daily for one week, and a growth curve is plotted. The maximum cell concentration should reach at least 10ⶠcells per ml.
**2. Cell Doubling Time Calculation**
Using the growth curve, the doubling time is calculated. The formula used is:
Doubling time = T / logâ‚‚(Y/X)
Where Y is the cell count at the peak, X is the initial number of seeded cells, and T is the time in hours before the peak. The doubling time for SP2/0 cells should not exceed 20 hours.
**3. Cloning Efficiency Determination**
Cells are diluted using the limiting dilution method and inoculated into 96-well plates at a density of 1 cell per well. At least 48 wells are used per plate. Cultures are maintained at 37°C with 5% CO₂ for one week. The cloning efficiency is calculated as:
Cloning rate = (A/B) × 100%
Where A is the number of wells with growing cells, and B is the total number of cells inoculated. The cloning rate should be no less than 70%.
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