Development and evaluation of a competitive enzyme-linked immunosorbent assay using a monoclonal antibody for diagnosis of severe fever with thrombocytopenia syndrome virus in bovine sera

Lee, Hyojin; Kim, Eun-Ju; Song, Jae-Young; Choi, Jeong Soo; Lee, Ji Youn; Cho, In-Soo; Shin, Yeun-Kyung

cELISA was performed as previously described, with slight modification [8] . Briefly, a Polysorp ELISA plate (Nunc, USA) was coated with 50, 100 or 200 ng/well of recombinant NP. The plate was then incubated for 16 hours at 4 o C to allow antigen binding. After washing with 0.5% Tween 20 (Sigma-Aldrich, USA) in 1× PBS to remove the unbound antigens, the plate was blocked with 5% skim milk and 1% horse serum in 1× PBS. For the serological test, 50 L of diluted test sera and the same amount of HRP-conjugated mAb (1 : 500) were added to the antigen coated plate and incubated for 90 min at 37 o C. The mAb was conjugated with HRP according to the instructions provided with the EZ-LinkTM Plus Activated Peroxidase kit (Thermo Fisher Scientific, USA). After washing the plate six times, 3,3',5,5'-tetramethylbenzidine peroxidase substrate (KPL) was added to develop the color. The reaction was terminated with 1 M sulfuric acid (Sigma-Aldrich). The optical density (OD) of the sample was calculated as follows: the OD at 450 nm was subtracted from the OD at 630 nm to adjust for the background absorbance. The OD values were converted to percent inhibition (PI) values using the following formula: PI = [1 − (OD test sample/OD negative control)] × 100.