Ultra 384 microplate reader

Two milliliter aliquots of urine were concentrated to 0.5 mL and centrifuged at 10000g for 10 min. Amicon ultra-0.5 mL centrifugal filters (3 kDa) purchased from EMD Millipore (Billerica, MA) were used to separate urinary proteins from small molecules according to the manufacturer’s protocol. Proteins captured on the filter were washed twice at 14000g for 30 min with water to remove possible interferences. Total protein concentration was measured from each sample via a Thermo Scientific BCA Protein Assay Kit (Rockford, IL) at an absorbance of 570 nm using a Tecan Ultra 384 microplate reader (Männedorf, Switzerland). Urinary protein samples were each normalized to 200 μg and further concentrated.

MERS-CoV neutralization assay was performed as previously described [43 (link),44 (link)]. Briefly, 293T cells in 10 cm² dishes were transiently co-transfected with a pcDNA3.1-MERS-CoV-S plasmid and a PNL4-3.luc.RE plasmid encoding an Env-defective luciferase-expressing HIV-1genome. After 48 h post-transfection, the produced pseudovirus was harvested from the supernatant, and filtered through 0.45 μm sterilized membrane. The MERS-CoV pseudovirus was incubated with four inhibitors at 37 °C for 30 min, and then pseudovirus and inhibitors were added to DPP4-expressing Huh-7 cells (10⁴/well) preplated in 96 well tissue culture plates for 6 h. After 12 h, fresh medium was added to the plates and incubated for another 48 h. Cells were lysed with lysis reagent (Promega, Madison, WI, USA) and lysates were transferred into 96-well Costar flat-bottom luminometer plates (Corning, Corning, NY, USA). Luciferase substrate was added and the readings were recorded with an Ultra 384 Microplate Reader (Tecan, Männedorf, Switzerland).