Bovine serum albumin (bsa)

The assay was performed on a 96-well plate coated with 1 µg of GFPE-N or GFPE-C antigen per well (20 ng/μL) and 100 mM carbonate buffer pH 9.6 as background control. Immobilization was carried out at 4 °C overnight. Subsequently, each well was blocked with 250 µL of 3% BSA (Sigma-Aldrich, Saint Louis, MO, USA) in PBS for 1 h at 37 °C. Thereafter, three washes with 0.05% Tween-20 in PBS (wash buffer) were performed. TEV or TEVK VLPs were mixed with anti-TEV antibody (Agdia, Elkhart, IN, USA) in 1:100 dilution using PBS/1% BSA as a diluent, and each mixture was incubated at 37 °C/200 rpm for 2.5 h. The antibody/VLP mixture was added to the wells in triplicate, calculating 1 µg of each VLP per well. As control, anti-TEV antibody alone was added in 1:100 dilution to antigen-coated wells (data not shown). To allow interaction, the plate was incubated at 37 °C for 1 h. Subsequently, the plate was washed three times with wash buffer and once with PBS. Finally, 50 µL of Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System for ELISA (Sigma-Aldrich, Saint Louis, MO, USA) was added to each well, and absorbance was read at 405 nm.