Hrp conjugated goat anti mouse igm or igg

SINV-specific IgM and IgG levels were evaluated in mouse sera by enzyme-linked immunosorbent assay (ELISA). Ninety-six well Maxisorp plates (Thermo Scientific Nunc, Marietta, OH) were coated overnight with a SINV TE-infected BHK cell lysate diluted in 50 mM NaHCO₃ (pH 9.6). Wells were blocked for two hours at 37°C with 10% FBS in PBS-0.05% Tween 20, and diluted serum samples were added and incubated overnight at 4°C. Sera were diluted 1:100 for IgM detection and 1:10 for IgG detection in 10% FBS in PBS-0.05% Tween 20. Antibody was detected with HRP-conjugated goat anti-mouse IgM or IgG (Southern Biotech, Birmingham, AL) diluted 1:1000 in 10% FBS in PBS-0.05% Tween 20, for one hour at 37°C and developed using BD OptEIA TMB Substrate Reagent Set (San Jose, CA) as substrate and 2N H₂SO₄ as stop solution. Absorbance was read at 450nm, and the resulting values were plotted as the optical density (OD) values of SINV-infected samples minus the OD values for uninfected control sera.

Anti-SINV antibody was measured in serum and 20% w/v brain homogenates in three to four mice per group per time point using an in-house enzyme immunoassay (EIA) as previously described (Baxter and Griffin, 2016 (link)). Briefly, Maxisorp 96-well plates (Thermo Scientific Nunc) coated with PEG-purified SINV TE were blocked in PBS-0.05% Tween-20 + 10% FBS for 2 h at 37°C and incubated overnight at 4°C with samples (1:100 [IgM] or 1:10 [IgG] for serum, 1:4 for brain homogenates, diluted in PBS-0.05% Tween-20 + 10% FBS). After incubating wells with 1:1000 HRP-conjugated goat anti-mouse IgM or IgG (Southern Biotech) for 2 h at RT, plates were developed using a BD OptEIA TMB Substrate Reagent kit and stopped using 2M H₂SO₄. Plates were read at 450nm and the optical density (OD) values for infected mice minus the OD values for mock-infected mice were graphed.
Interferon-gamma (IFN-γ) production was quantified in 20% w/v brain homogenates in 3-5 mice per group per time point by commercial EIA kit (Ebioscience Ready-SET- Go!). Assays were performed according to manufacturer's instructions, and data are presented as pg per gram brain. The standard curve for the assay ranged from 312.5 to 20,000 pg / g.