Horseradish peroxidase labeled anti mouse antibody

A set of 38 overlapping peptides of 15-mer peptides with an offset of two amino acids spanning domain D/A (residues 780–868) of the CSFV TD/96 strain was synthesized (Mimotopes Pty Ltd, Australia). Each mAb (diluted 1:400 in 2%BSA/PBS containing 0.1% sodium azide) was added to each well of the plate with the individual peptides and subjected to shaking and incubation at 20 °C for 1 h. Afterwards, the wells were washed four times with PBS containing 0.1% (v/v) Tween 20 (PBS/Tween 20). Bound antibodies were detected by reaction at 20 °C for 1 h using a conjugate solution that contained a horseradish peroxidase labeled anti-mouse antibody at a dilution of 1:2000 (0.5 μg/mL) (Thermo Fisher Scientific) in 2% BSA/PBS. Wells were washed four times with PBS/Tween 20, followed by two washes with PBS, and subsequently incubated at 20 °C for 45 min with 1-Step™ ABTS (Thermo Fisher Scientific). The absorbance was measured by a plate reader in the dual-wavelength mode at 405 nm against a reference wavelength of 492 nm.