LV-Based ELISA

An ELISA was used to detect anti-VesG antibodies in murine sera. For this, LVs pseudotyped with different VesGs and with no envelope (ΔEnv) were produced as previously described. Total protein concentrations of the LV preparations were determined using a Pierce BCA Protein Assay Kit, according to the manufacturer’s instructions, using BSA as the standard. A coating mix of 25 μg/mL total protein in PBS was prepared, and each well of Nunc Maxisorp ELISA plate (Thermo Fisher Scientific, UK) was coated at a volume of 100 μL/well overnight at 4°C. The plate was washed three times with 200 μL PBS before the samples were incubated with 200 μL/well blocking buffer and 2% fish gelatine (Sigma-Aldrich) in PBS, for 1 h at 37°C. The plate was washed three times with 300 μL/well washing buffer, PBS-0.05% (v/v) Tween20, before 100 μL/well serum samples diluted in diluent buffer, 10% fetal calf serum (FCS) (heat inactivated) in PBS, were added to the wells and incubated at 37°C for 2 h. After another three washes, the samples were incubated with the secondary antibody, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) diluted 1:1,000 in diluent buffer for 1 h at 37°C. Following three washes, 100 μL/well Ultra TMB-ELISA Substrate (Thermo Fisher Scientific) was added to each well incubated at room temperature for 10 min, and the reaction was stopped by adding an isovolume of 1 M sulfuric acid. The absorbance was determined at 450 nm using a FluoStar Omega Plate Reader (BMG Labtech).