Anti mouse igg coupled with horse radish peroxidase

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).

ELISA antigen was prepared by transfecting 293-T cells with pCAGGS expression plasmids either expressing no foreign protein (control antigen) or KFDV prM and E. At 24 h post transfection, cells were lysed with RIPA buffer (Thermofisher scientific, Waltham, MA, USA) and diluted in PBS. Purified EBOV GP was used as an EBOV antigen^{66 (link)}. ELISA plates (96-well flat bottom, NUNC, Waltham, MA, USA) were coated with 100 µl of the antigens at 4 °C overnight and blocked for 1 h at room temperature with 5% powdered milk in PBS and 0.05% Tween 20 (Fisher Scientifc) (PBST). Subsequently, serial dilutions of mouse sera in PBST were added to the plate and incubated for 1 h at room temperature. Detection was performed using anti-mouse IgG coupled with Horse Radish Peroxidase (Jackson ImmunoResearch) for 1 h at room temperature followed by adding ABTS substrate solution (Seracare) for 15 min at room temperature. Plates were read at 405 nm using ELISA reader. To obtain the test result the OD₄₀₅ of the control antigen coated wells was subtracted from the that of the KFDV antigen coated wells.