Evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of Middle East respiratory syndrome coronavirus among livestock in Ethiopia

TERAMICHI, Takurou; FUKUSHI, Shuetsu; HACHIYA, Yuma; MELAKU, Simenew Keskes; OGUMA, Keisuke; SENTSUI, Hiroshi

Synthetic S1 fragment of MERS-CoV was obtained from Sino Biolobical Inc. (Beijing, China) and used as the antigen [17] . ELISA microplates were coated with 50 µl of 50 ng/ml antigen per well at 4°C overnight, following which the wells were incubated with PBS containing 2% Block Ace and 0.05% Tween 20 for 2 hr at 37°C. Following the removal of blocking reagent, diluted serum samples were added and incubated for 1 hr at 37°C. After washing the wells thrice with 0.05% Tween 20 in PBS (PBS-T), a peroxidase-labeled protein AG (Thermo Fisher Scientific, Waltham, MA, U.S.A.) was added and incubated for 1 hr at 37°C. Following further washing thrice with PBS-T, 100 µl of 2,2′-azinobis-3-ethylbenzthiazolinesulfonic acid (ABTS) substrate solution (Roche Applied Science, Penzberg, Germany) was added and incubated for 20 min at room temperature. The optical density (O.D.) of each well was measured at 450 nm using a microplate reader, and mean absorbance was determined for each serum sample. One of camel serum that showed a high antibody titer in the neutralization test by live MERS-CoV was treated as a positive control.