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LC03-3.3 SOP8 TVS Static Protection 3.3V
First, determine the serotype of the target virus. Next, prepare all necessary experimental materials. These include:
1. **Patient samples**: Serum from individuals in the recovery phase of hemorrhagic fever.
2. **Materials**:
- Hantavirus standard strain 76-118 and Seoul virus standard strain.
- Rabbit anti-Hantavirus and anti-Seoul virus serum.
- Reagents commonly used in plaque reduction neutralization tests (PRNT).
**Procedure**:
1. Dilute the test serum with bovine serum Hanks’ solution to a 1:10 ratio and inactivate it at 30°C for 30 minutes.
2. Further dilute the serum to 1:20, 1:40, 1:80, etc., while keeping the control serum at 1:10.
3. Dilute both viruses (in an ice bath) to a concentration of 200 PFU/ml.
4. Mix each serial dilution of the serum with 200 PFU/ml of each virus solution (0.3 ml each), incubate at 37°C for 1 hour (shaking every 15 minutes).
5. Remove the maintenance solution from each well in the cell culture plate.
6. Inoculate Vero-E6 cells in a 24-well plate with the serum-virus mixtures, standard serum control, and virus controls. Use two wells per dilution, adding 100 µl per well. Incubate at 37°C for 1 hour, shaking every 15 minutes.
7. Add the first agarose overlay (around 42°C) at 1 ml per well, allow it to solidify at room temperature, then incubate at 37°C in a 5% CO₂ incubator for 7–9 days.
8. Add the second agarose overlay containing neutral red (1 ml per well). Solidify at room temperature, place in a 37°C, 5% CO₂ incubator for 2–5 days, and observe plaques starting the next day.
9. Determine the antibody titer by identifying the highest serum dilution that reduces plaque formation by 50% or more compared to the virus control.
10. **Serotype determination**: Compare the antibody titers against Hantavirus and Seoul virus. If the titer against Hantavirus is at least four times higher than that of Seoul virus, classify as Hantavirus type. Conversely, if the titer against Seoul virus is higher, classify as Seoul virus. If the titers are similar, classification may not be possible.
**Preparation of Agarose Solutions**:
1. First agarose overlay: Prepare as needed.
2. Second agarose overlay: Similar to the first, but replace inactivated fetal bovine serum with 5 ml, and add 1 ml of neutral red solution (333.0 µg/L neutral red sodium salt from Gibco).
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