Sensitive Virus Detection by Sandwich FLISA

Sandwich FLISA was performed as described previously [29 (link)]. Briefly, a black 96-well microtiter plate (Greiner GmbH) was coated with 100 µL/well of 10 µg/mL antibody candidate in coating buffer (pH 9.6) and incubated at 4 °C for 12 h. After 5% non-fat dry milk blocking at 37 °C for 2 h, the recombinant virus (100 µL/well) was added and the plate was incubated for another hour at 37 °C. After washing the plate with 200 μL PBS-T (pH 7.4), Eu nanoparticle (NP)-conjugated antibody (100 μL/well) was added and the plate was incubated at 37 °C for another hour for antigen detection. Stringent washing with PBS-T (pH 7.4) was performed five times to remove nonspecific binding, and 100 μL PBS was added to each well. Fluorescence was then measured in the Infinite F200 microplate reader (TECAN, Männedorf, Switzerland) at 355 nm (excitation) and 612 nm (emission) wavelengths.