Specificity of Anti-MERS-CoV S1 Nanobodies

The specificity of anti-S1^A, anti-S1^B, and control nanobodies for MERS-CoV S1, S1^A, and S1^B proteins was determined using ELISAs as previously described (29 (link)). In brief, 96-well ELISA plates were coated with 1 μg/ml MERS-CoV S1 (amino acids 1 to 751), S1^A (amino acids 1 to 357), or S1^B (amino acids 358 to 588) protein in phosphate-buffered saline (PBS) (pH 7.4) and incubated overnight at 4°C. Wells were then washed with PBS and blocked with 1% bovine serum albumin in PBS–0.5% Tween 20 for 1 h at 37°C. Nanobodies were 2-fold serially diluted in blocking buffer starting at a 1-μg/ml concentration, 100 μl of each dilution was added per well, and plates were incubated at 37°C for 1 h. Next, plates were washed three times in PBS–0.05% Tween 20 (PBST), after which they were incubated with mouse anti-His tag antibodies (1:2,000; Thermo Fisher Scientific) at 37°C for 1 h. Following incubation, the plates were washed and further incubated with goat anti-mouse HRP (1:2,000; Dako) at 37°C for 1 h. After this incubation, plates were washed three times in PBST, a 3,3′,5,5′-tetramethylbenzidine substrate (eBioscience) was added, and the plates were incubated for 10 min. The reaction was stopped with 0.5 N H₂SO₄ (Sigma). The absorbance of each sample was read at 450 nm with an ELISA reader (Tecan Infinite F200).