2.4 Enzyme-linked immunosorbent assay (ELISA)
For sandwich ELISA, 96-well microplates were coated with mouse monoclonal anti-rabies virus (RV1C5, Santa Cruz) at 1 μg/ml concentration in carbonate/bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C. The plates were blocked with 5% non-fat dry milk in PBS at room temperature (RT) for 2 h and then with serially diluted VSV/RABA-GP at RT for 2 h. After four washes with PBS-T, the plates were incubated with horseradish peroxidase (HRP) conjugated anti-rabies virus (Rab-50) at RT for 1 h.
For indirect ELISA, ELISA plates were coated with inactivated RABV in carbonate/bicarbonate buffer (pH 9.6) overnight at 4 °C. The plates were blocked with 5% non-fat dry milk in PBS at RT for 2 h and then with serially diluted mouse sera at RT for 2 h. After four washes with PBS-T, the plates were incubated with HRP conjugated anti-mouse IgG, IgM, IgG1 or IgG2a at RT for 1 h. The reaction was visualized by substrate 3,3‘,5,5‘-tetramethylbenzidine and stopped with 1N H2SO4. The absorbance at 450 nm was measured by ELISA plate reader.