Rsv g protein

RSV A2 virus-specific antibodies (IgG, IgG1, and IgG2a) were determined in sera by enzyme-linked immunosorbent assay (ELISA) using FI-RSV (4 μg/ml) [14 (link)], RSV F protein (100 ng/ml, BEI, NIAID, NIH), or RSV G protein (200 ng/ml, Sino biological Inc.) as a coating antigen. RSV-specific neutralizing antibody titers in mouse sera were measured using the red fluorescent monomeric Katushka 2 protein expressing RSV A2 strain (A2-K-line19F) as described [21 (link), 22 (link)]. Equal volumes of the diluted sera were mixed with A2-K-line19F virus to yield approximately 100 red-fluorescent (excitation at 588 nm, emission at 635 nm) RSV infected-cell spots per well. The percentages of red-fluorescent RSV infected-cells were presented as a measure of serum neutralization activity.
RSV titers in lung samples at day 5 post-challenge were determined using an immunoplaque assay as described [14 (link)]. Serially diluted lung homogenates were added to the HEp2 cell monolayer plates and incubated 3–6 days at 37°C. After fixing with ice-cold acetone-methanol and air drying, individual plaques were visualized and counted by anti-RSV F monoclonal antibody, HRP conjugated anti-mouse IgG antibody, and 3,3′-Diaminobenzidine tetrahydrochloride substrate (Invitrogen). The viral load detection limit is estimated to be approximately 60 PFU from each mouse lung sample in this assay.

To determine antigen-specific antibody levels, maxisorp immunoplates were coated with FI-RSV (4 μg/ml), RSV F protein (100 ng/ml, BEI, NIH) or RSV G protein (200 ng/ml, Sino biological Inc.) as an ELISA antigen. RSV-specific neutralizing antibody titers in mouse sera were measured using the red fluorescent monomeric Katushka 2 protein expressing RSV A2 strain (A2-K-line19F) ^{18 (link),19 (link)} as detailed in the supplementary materials.