TABLE 2.
CONSISE consensus 3-day HA MN assay for detecting antibodies to A(H1N1)pdm09 virus
| Parameter | Required parameter | Recommended parameter |
|---|---|---|
| Stock virus prepn | ||
| Cell substrates for virus growth | Day 10 embryonated eggs, MDCK cells, MDCK-SIAT1 cells | |
| Stock virus infectivity concn and method of determination | At least 106 TCID50/ml, read by RBC agglutination | |
| Stock storage | Aliquots of bulk virus prepn | |
| Serum prepn | ||
| Storage of sera following receipt | −70°C, −20°C, and 4°C, 1 or 2 freeze-thaw cycles in testing laboratory | |
| Preassay treatment of sera | Heat treatment at 56°C for 30 min, without dilution in media | |
| Initial serum dilution | 1:10 | |
| Sample type | Sera only or plasma only | |
| Virus prepn | ||
| Final virus amt per well | 100 TCID50 | |
| Vol of virus solution added per sample/well | 50 μl, 100 μl, or 200 μl | |
| Virus/serum mix incubation | 1 h at 37°C | |
| Virus/serum mix incubation on cell monolayer | 1 h at 37°C | |
| Calculated starting serum dilution | 1:10, excluding virus vol | |
| Cell prepn | ||
| Prepn of cells | Preformed monolayer | |
| Cell types used | MDCK (ATCC), MDCK (Salisbury), MDCK-SIAT1 | |
| Assay diluent | Coon's/Dulbecco's modified Eagle's medium, with trypsin (1/2 μg/ml), laboratory-preferred media | |
| Cell infection media | Coon's/Dulbecco's modified Eagle's medium, with trypsin (1/2 μg/ml), laboratory-preferred media | |
| Assay setup | ||
| Incubation time of assay to endpoint reading | 3 days | |
| Incubation conditions | 35–37°C, 5% CO2 | |
| No. of sample replicates | Replicates preferred if available | |
| Endpoint estimation | ||
| Endpoint determination | Turkey/guinea pig RBC agglutination, CPE | |
| Endpoint calculation method | 50% neutralization |