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LC03-3.3 SOP8 TVS Static Protection 3.3V
First, determine the serotype of the virus under investigation. Second, prepare all necessary experimental materials. These include:
1. Serum samples collected from patients during the recovery phase of hemorrhagic fever.
2. Experimental reagents:
- Hantavirus standard strain 76-118 and Seoul virus standard strain.
- Rabbit anti-Hantavirus and anti-Seoul virus sera as standard controls.
- Reagents commonly used in plaque reduction neutralization tests (PRNT), such as cell culture media, bovine serum, and agarose.
Third, follow these steps for the test:
1. Dilute the test serum with Hanks’ balanced salt solution containing bovine serum to a 1:10 dilution, then 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. Prepare both viruses by diluting them on ice to a concentration of 200 PFU/ml.
4. Mix each serum dilution (0.3 ml) with 200 PFU/ml of each virus, incubate at 37°C for 1 hour, shaking every 15 minutes.
5. Remove the maintenance solution from each well of a 24-well plate.
6. Inoculate Vero-E6 cells with the serum-virus mixtures, standard serum control, and virus-only controls. Use two wells per dilution, with 100 μl per well. Incubate at 37°C for 1 hour, shaking every 15 minutes.
7. Add the first agarose overlay (approximately 42°C) at 1 ml per well, let it solidify at room temperature, and incubate at 37°C in a 5% CO₂ incubator for 7–9 days.
8. Add a second agarose layer containing neutral red (1 ml per well). Let it solidify at room temperature, then place the plate back into the incubator for 2–5 days. Observe plaque formation the next day.
9. Calculate the antibody titer based on the highest dilution that reduces plaque formation by 50% or more compared to the virus control.
10. Determine the virus type by comparing the titers against Hantavirus and Seoul virus. If the titer against Hantavirus is at least four times higher than that against Seoul virus, classify it as Hantavirus. Conversely, if the Seoul virus titer is higher, classify it as Seoul virus. If the titers are similar, classification may not be possible.
Fourth, prepare the agarose overlays:
1. First agarose layer: Typically composed of agarose, cell culture medium, and fetal bovine serum.
2. Second agarose layer: Similar to the first, but with the addition of 5 ml inactivated fetal bovine serum and 1 ml of neutral red solution (333.0 µg/L sodium salt of neutral red, Gibco).
This detailed procedure ensures accurate detection and classification of hantaviruses, providing critical information for diagnosis and epidemiological studies.
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