Mrx11 plate reader

Blood samples were collected immediately from mice by cardiac puncture and left at room temperature to clot. Parasite-specific and VLP recombinant protein-specific antibodies (IgM, IgG1 and IgG2c) were determined in sera by enzyme-linked immunosorbent assay (ELISA) as previously described [91 (link)]. Briefly, 96-well plates were coated with 50 μg/well of the overnight T. muris E/S antigen at 5 μg/ml or with 50 μg/well of the purified VLP recombinant protein in 0.5 M carbonate-bicarbonate buffer for overnight at 4°C. The plates were washed and then blocked with 3% BSA (Sigma-Aldrich) in PBS Tween-20 (PBS-T20) (0.05% Tween 20, Sigma-Aldrich) for 1 h at 37°C. After washing, diluted sera were added and incubated for 1 h at 37°C. Antibody responses were detected using the Biotinylated rat anti-mouse IgM, IgG1 and IgG2a/c (BD Biosciences). After washing, streptavidin peroxidase (Sigma-Aldrich) was added to the plates and incubated for 1 hour at room temperature. The TMB ELISA substrate (3, 3’, 5, 5’-tetramethylbenzidine- Thermo) was used to develop color and stopped with 0.003% (H₂SO₄). The optical density was measured with a Dynex MRX11 plate reader (DYNEX Technologies) at 450 nm with a reference of 570 nm.

T. muris specific IgG1 and IgG2a antibody responses were investigated in the serum of day 19 infected mice. ELISA plates were coated with 5 μg/ml T. muris ES antigen diluted in carbonate bicarbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃ adjusted to pH 9.6) and incubated overnight at 4 °C. Plates were washed with PBS Tween (PBS containing 0.05% Tween20, Sigma-Aldrich) and blocked with 3% BSA in PBS Tween. Serum was diluted 1:20–1:2560 in PBS Tween, added to the plate at 50 μl/well, and incubated for 1 h at 37 °C. Plates were washed and incubated with 50 μl/well of biotinylated rat anti-mouse IgG1 (BD Biosciences, Oxford, UK) or IgG2a (BD Biosciences) for 1 h at room temperature. After washing, plates were incubated with 75 μl/well streptavidin β peroxidase (Roche Diagnostics GmbH, Germany) for 1 h at room temperature. Plates were then washed and developed with 100 μl/well of 3,3′, 5,5′ tetramethylbenzidine (Ultra TMB ELISA substrate, Thermo Fisher Scientific). The color development was stopped by adding 100 μl/well of 2 N sulphuric acid (R&Dsystems, Abingdon, UK). Absorbance was read using a Dynex MRX11 plate reader (Dynex Technologies, West Sussex, UK) at 405 nm with a reference of 490 nm.