Igg2c

Serum of T. muris infected mice was analyzed for parasite specific IgG1 and IgG2c antibody levels. First, 96 well plates were pre-coated with 5µg/ml T. muris overnight E/S antigen overnight. After washing, non-specific binding was blocked using 3% BSA (Sigma-Aldrich) in PBS. Post blocking and subsequent washing, the plates were incubated with the serum samples at 2 fold dilutions ranging from 1:20 to 1:2560. The parasite specific antibody was detected using biotinylated IgG1 (BD Biosciences) or IgG2c (BD Biosciences) antibodies together with Streptavidin-POD (Roche). After washing, the plates were developed with the TMB substrate kit (BD Biosciences, Oxford, UK) following the manufacturer’s instructions. Upon sufficient coloration, the reaction was stopped using 0.18 M H₂SO₄. Absorbance was measured at 450 nm, with reference of 570 nm subtracted, with a Versa max microplate reader (Molecular Devices) with SoftMax Pro 6.4.2.

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).

Serum was assayed for parasite specific IgG1 and IgG2c antibody production. 96 well plates were coated with 5μg/ml T. muris overnight E/S antigen overnight, plates were washed, and non-specific binding blocked with 3% BSA (Sigma-Aldrich) in PBS. Following washing, plates were incubated with serum (2 fold dilutions, 1:20–1:2560) and parasite specific antibody was measured using biotinylated IgG1 (BD Biosciences) or IgG2c (BD Biosciences) antibodies which were detected with SA-POD (Roche). Finally, plates were washed and developed with TMB substrate kit (BD Biosciences, Oxford, UK) according to the manufacturer’s instructions. The reaction was stopped using 0.18 M H₂SO₄, when sufficient colour had developed. The plates were read by a Versa max microplate reader (Molecular Devices) through SoftMax Pro 6.4.2. software at 450 nm, with reference of 570 nm subtracted.