Abts substrate solution

Each mAb was diluted in PBS to a concentration of 2.5 μg/mL, and then added to an ELISA plate (clear or black) (Thermo Fisher Scientific, Rockford, IL, USA). After blocking with 2% skim milk, 100 μL of antigen protein (2 ng/mL) diluted with PBS-T or blank (PBS-T alone) was added and incubated for 60 min at RT. After three washes with PBS-T, 100 μL of each mAb conjugated with horseradish peroxidase (HRP) was added into each well and incubated for 60 min at RT. After washing with PBS-T, 100 μL of ABTS substrate solution (Kirkegaard & Perry Laboratories, Washington, D.C., USA) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, IL, USA) was added and incubated. Absorbance at 410-490 nm or luminescence was measured on a GloMax Explorer plate reader (Promega, WI, USA), and the signal-to-noise ratio (S/N) was calculated.

Each mAb was diluted in 50 mM of carbonate buffer (pH 9.6) to a concentration of 10 μg/mL, and then added to an ELISA plate (AGC TECHNO GLASS, Shizuoka, Japan). To immobilize the antibodies, the plate was incubated overnight at 4°C. Wells were blocked with PBS containing 2% (w/v) skim milk for 1 h at room temperature (RT). After three washes with PBS containing 0.05% (v/v) Tween-20 (PBS-T), 100 μL of antigen protein (1 ng/mL) diluted with PBS-T or blank (PBS-T alone) was added and incubated for 60 min at RT. After three washes with PBS-T, 100 μL of each mAb conjugated with horseradish peroxidase (HRP) was added into each well and incubated for 60 min at RT. Antibody labeling was performed using the Peroxidase Labeling Kit -NH₂ (Dojindo Laboratories, Kumamoto, Japan). After three washes with PBS-T, 100 μL of ABTS substrate solution (Kirkegaard & Perry Laboratories, Washington, DC, USA) was added and incubated for 30 min at RT. Absorbance at 415/492 nm was measured on a plate reader, and the signal-to-noise ratio (S/N) was calculated.

ELISA antigen was prepared by infecting VeroE6 cells with AHFV strain 2003 and collecting supernatant at 48 h post infection. Following low-speed centrifugation, AHFV was pelleted by ultracentrifugation, resuspended in PBS + 2% Triton-X 100 and irradiated with 10 megarads [26 (link)]. ELISA plates (96-well flat bottom, maxisorp, NUNC, Waltham, MA, USA) were coated with 100 µl of the antigens (1:1000 dilution in PBS) at 4°C overnight and blocked for 1 h at room temperature with 5% powdered milk in PBS and 0.05% Tween 20 (Fisher Scientific) (PBST). Subsequently, serial dilutions of mouse sera in PBST were added to the plate and incubated for 1 h at room temperature. Convalescent serum from mice infected with KFDV was used as a positive control. Detection was performed using anti-mouse IgG coupled with horse radish peroxidase (Jackson ImmunoResearch) for 1 h at room temperature followed by ABTS substrate solution (Seracare) for 15 min at room temperature. Plates were read at 405 nm using an ELISA reader (BioTek Instruments).