Assay diluent

NJ85 peptide and ovabumin were dissolved in ELISA Coating Buffer (eBioscience) and coated (2µg/well) onto a 96-well ultra-clear polypstyrene microtiter plate overnight at 4°C. The plate was washed with wash solution (1X PBS and 0.05% Tween 20) and blocked with 1x assay diluent (eBioscience) for 1h at room temperature. After four washes, 100µl of diluted serum was added and incubated overnight at 4°C. Subsequently, after five washings, 100µl of 1/5000 diluted biotin-conjugated anti-rabbit IgG (KPL Inc.) was added, and the mixtures were incubated for 1h. After washes, 100µl/well of 1/500 diluted Avidin-HRP (eBioscience) were added and incubated for 30min. After four washes, 100µl of TMB (eBioscience) was added to each well for colar development. The reaction was stopped by adding of 50µl of 2N H₂SO₄. The absorbance was determined at 450nm using an Envision Alpha Multilabel Reader (PerkinElmer).

Blood samples were collected from LRV1+ Lg or LRV1- Lg infected mice 8 weeks after infection. Once coagulated, samples were centrifuged at maximum speed and serum was collected and kept at -20°C until analysis. ELISA against LRV1c was performed on the serum to quantify the level of specific IgGs against the viral capsid. Briefly, ELISA plates (Maxisorp, Nunc) were coated with 5μg/mL LRV1c in PBS O/N at 4°C. The plate was then washed with PBS-Tween (0.05% Tween) and blocked with 1x assay diluent (eBioscience) for 1 hour at RT.
Sera from LRV1+ Lg, LRV1- Lg or uninfected control mice were then plated in a 3 fold serial dilution with a starting concentration of 1:50. After 2h of incubation and washes with PBS- Tween, a secondary antibody against total IgGs (Jackson Immunoresearch, goat anti-mouse, 1:5000), IgG2c (rat anti-mouse, 1:3000, BD bioscience) or IgG1 (rat anti-mouse, 1:5000, BD bioscience) coupled with Biotin was added to the plate and incubated for 1 hour at RT before adding during 20 min streptavidin-HRP (1:250, eBioscience). Quantification was determined by colorimetric assay using TMB substrate according to supplier’s instructions (eBioscience).