Table 1.
Assay Overview Comparison. Comparison between each viral titering method. Each assay type presents a fundamentally different method of determining viral titer, and requires differences in time and sample volume. Plate formats may vary. For the purposes of this paper, the FFU assay was done in a 384 well format. Methods for use in a 96 well format have been included in this paper.
| Parameter | NRAO | CVMO | TCID50 | FFU |
|---|---|---|---|---|
| Plate Format | 6 Well | 6 Well | 96 well | 384 well (Can be done in other formats including 96 well) |
| Volume of inoculum | 100 µL | 100 µL | 20 µL | 25 µL (384 well) 20 µL (96 well) |
| Challenge Incubation | 1 h | 1 h | 10 days | 2 days |
| Overlay/staining solution | Primary: 1:1 mixture of buffered EMEM and Agarose Secondary: 1:1 mixture of buffered EMEM containing Neutral Red (stain) and Agarose |
1:5 mixture of buffered DMEM and MethylcelluloseStained with crystal violet | Stained with crystal violet | Immunofluorescent staining using virus-specific antibody |
| Time to complete | 8 days | 8–10 days | 8–10 days | 3 days |
| Read out method | Plaque count, manual | Plaque count, manual | Gross cell death within replicates, manual | Automated image analysis |
| Titer Calculation | Plaque count in well of lowest dilution, dilution, total volume in well | Plaque count in well of lowest dilution, dilution, total volume in well | Reed–Muench calculation | Number of infection foci stained with fluorescent antibody in lowest dilution, dilution, total volume in well |
| Estimated Hands-on Time, Hours (For 40 Test Points) | 10 | 10 | 8 | 5 |