| Viral neutralization, infection of cells and replication |
| Samples collection |
Collection of blood sample by venipuncture. Inactivation of separated sera at 56°C for 30 min. |
| Dilution of sera |
Preparation of seven 3-fold dilution steps in a flat-bottom 96-well plate in a final volume of 100 ul. Preparation of 150 µl medium for non-neutralized virus control. A control serum with established NT titer should be included in each assay plate as a standard. |
| Administration of virus and neutralization |
Dispensing of 50 µl containing 400 pfu of VTT to each well. Incubation at 37°C, 5% CO2 for 1 h. |
| Addition of cells and viral replication |
Addition of 100 µl containing 30,000 Vero cells to each well. Incubation at 37°C, 5% CO2 for 24 h. |
| Luciferase assay |
| Addition of substrate |
Aspiration of 100 µl supernatant. Addition of 100 µl/well of Bright-GloTM luciferin equilibrated to room temperature. Incubation at room temperature (20°C) for 2 min, keeping from light. |
| Measurement of luminescence |
Measuring luminescence with the GLOMAXTM 96 microplate luminometer. |
