Nunc immuno maxisorb plates

Antigen integrity Nunc-Immuno Maxisorb™ plates (ThermoFisher Scientific, Waltham, MA, USA) coated with capture monoclonal anti-clamp IgG2a at 4 µg/mL were incubated for 2 hours with PBS containing 2% bovine serum albumin (BSA) (w/v) (Sigma, MO, USA), washed (PBS containing 0.05% Tween20 [v/v]), and incubated with dilutions of adjuvanted formulations for 1 hour at 37°C. Plates were then washed twice and incubated for 1 hour with the human anti-MERS m336 monoclonal antibody (43 (link)). Plates were then washed and incubated with a polyclonal goat anti-human IgG coupled to peroxidase (Sigma A0170, MO, USA). Plates were then washed 4 times before addition of o-phenylenediamine dihydrochloride (OPD) peroxidase substrate. reaction was stopped with the addition of a 25% sulfuric acid (v/v) solution. Absorbance was measured at 492 nm using a microplate reader (Tecan, Switzerland).

Anti-MERS Ig responses were measured in mouse sera using ELISA. Nunc-Immuno Maxisorb™ plates (ThermoFisher Scientific, MA, USA) were coated directly with MERS SClamp antigen at 0.25 µg/mL in PBS overnight at 4°C, blocked for 2 hours with PBS containing 2% BSA (w/v) (Sigma, MO, USA), washed with PBS containing 0.05% Tween20 (v/v), and incubated with 4x serial dilutions of mouse serum samples diluted 1/400 in PBS containing 0.05% BSA. Plates were incubated for 90 minutes, washed, and further incubated for 1 hour with anti-mouse antibodies coupled to HRP (SouthernBiotech, AL, USA) total Ig, IgG1, IgG2b or IgG2c. Plates were then washed before addition of 3, 3’, 5, 5’-Tetramethylbenzidine (Sigma, MO, USA) for 4 minutes and the reaction was stopped by adding 1 M sulfuric acid. Absorbance was then measured at 450 nm using a microplate reader (Tecan, Switzerland). The calculation of percentages shown in Figure 2D was based on the values displayed in Figures 2A–C. Percentages were calculated using the following % = 100 x (m/b) where m = the group average for either IgG1, IgG2b, or IgG2c, and b = the sum of each group’s average (IgG1 + IgG2b + IgG2c).