*2.8. Detection of SUDV GP-Specific Antibody by ELISA*

The mice and horse serum samples were collected two weeks after each immunization. Levels of SUDV GP-specific antibodies were detected by indirect ELISA. Briefly, 100 μL of purified -prokaryotic expressed SUDV GP was coated on ELISA plates at a concentration of 10 μg/mL overnight at 4 ◦C, and then the plate was blocked with 2% BSA for 2 h at 37 ◦C. The serum was added (100 μL/well), and it was 2-fold serially diluted in 2% BSA; plates were subsequently incubated for 1.5 h at 37 ◦C, and 100 μL of HRP-labeled goat anti-mouse IgG or HRP-labeled goat anti-horse IgG (Bioss antibodies, Beijing, China), diluted 1:10,000 in 2% BSA was added to each well. After one hour of incubation at 37 ◦C, 100 μL of 3,3 ,3,5 -tetramethylbenzidine (TMB) (Sigma-Aldrich, St. Louis, MO, USA) substrate solution was added to each well and then was stopped with the addition of 50 μL of 0.5 M H2SO4. Optical density (OD) values were measured at a wavelength of 450 nm (OD450). After each incubation step, ELISA plates were washed five times with PBST. Mean OD values were considered positive if the OD values were more than two times the value of the negative control.
