2.10. Sandwich ELISA

Sandwich ELISA was carried out according to Panferov et al. [33] with the following modifications: the captured antibodies were used at a concentration of 1 μg∙mL⁻¹ in PBS; the viruses were at concentrations ranging from 0.45 ng∙mL⁻¹ to 1000 ng∙mL⁻¹; the biotinylated antibodies were at a concentration of 1 μg∙mL⁻¹. After the completion of all of the stages, including the addition of streptavidin–polyperoxidase conjugate, TMB substrate, and stop solution (1 M H₂SO₄), the optical density at 450 nm (OD₄₅₀) was measured.

The enzyme-linked immunosorbent assay (ELISA) was performed using a Thermo Electron WellWash 4 MK2 washer (Fisher Scientific, Hampton, NH, USA). The results were registered by a Zenyth 3100 microplate reader (Anthos Labtec Instruments, Salzburg, Austria). All the statistical processing and calculations were done using Origin Pro 9.0 (Origin Lab, Northampton, MA, USA). The LOD was determined using the three-sigma method. The cross-reactivity was calculated as the ratio between the midpoints (IC50) of the calibration curves of PVY^N and the cross-reactant (i.e., another virus) multiplied by 100%.