RSV Antigenicity Conservation Examination

To examine the conservation of RSV antigenicity, dot blot analysis and enzyme-linked immunosorbent assays (ELISAs) were performed. ELISAs were performed as described with the same antibodies as dot blots (19 (link)–21 (link)). For dot blot, 2 µL of each inactivated RSV sample, with respective untreated controls, were put on a nitrocellulose membrane (GE Healthcare Life Sciences, Germany). The membranes were blocked with 5% (w/v) skimmed milk powder (Carl Roth, Germany) in PBS-T (PBS with 0.05% Tween 20) (Bio&Sell, Germany; Carl Roth) for 1 h and incubated with a monoclonal antibody (mAb) recognizing RSV-F [18F12, 1:400 (26 (link))], a mAb recognizing the prefusion form of RSV-F (RSV-preF) (D25, 1:1,000; Cambridge Biologics, USA), or a mAb recognizing RSV-G (8C5, 1:500; Invitrogen, USA) in 2% (w/v) skimmed milk in PBS-T at 4°C overnight. After washing three times with PBS-T, the membranes were incubated with the respective secondary antibody: for 18F12 and 8C5, the peroxidase AffiniPure™ sheep anti-mouse IgG (H+L)-horseradish peroxidase (HRP) antibody (Dianova, Germany) diluted 1:500, and for D25, the goat anti-human IgG HRP-conjugated 1:20,000 (Dianova) for 1 h. The membranes were washed, developed with enhanced chemiluminescent substrate (Pierce, USA), and imaged on a CELVIN^® chemiluminescent imager (Biostep, Germany).