Separate 96 well plates

Based on the antigen used for immunizations, NP conjugated to BSA at a >20 ratio (Biosearch Technologies), PE or OVA all diluted in PBS were coated onto separate 96 well plates (Greiner Bio-one) at 2 μg/mL and incubated overnight at 4 ^°C. Plates were washed then blocked with casein (Thermo Scientific) for one hour at room temperature (RT). Serum samples were serially diluted in casein then added to the plates and incubated for two hours at RT. After washing, biotin rat anti-mouse IgG1 (BD), peroxidase conjugated goat anti-mouse IgM (Jackson Immunoresearch) or biotin rat anti-mouse IgE (BD) was added to the plates and incubated for one hour at RT. Plates were washed and streptavidin HRP (Jackson Immunoresearch) was added to the IgG1 and IgE plates and incubated for thirty minutes at RT. Plates were washed then developed for twenty minutes with an o-Phenylenediamine dihydrochloride tablet (Sigma) dissolved in 100 mM Sodium phosphate dibasic (Mallinckrodt), 50 mM citric acid (Mallinckrodt) and 0.04% hydrogen peroxide (Mallinckrodt) at pH 5.0. Reactions were stopped using 1N HCL (Fisher Scientific) and plates were read at 490λ using a Molecular Devices Spectra Max 340 PC running Soft Max Pro 3.1.2. All antibody data are presented at IgM (1:1000), IgG1 (1:80,000), and IgE (1:100) serum dilutions which represent non-saturating concentrations.

Separate 96 well plates (Greiner Bio-one) were coated with calf thymus DNA (R&D) at 10 μg/mL and incubated overnight at 4°C. Plates were washed with D-PBS (Thermo Scientific) then blocked with D-PBS containing 2% bovine serum albumin fraction V (Thermo Scientific) for 1 h at room temperature (RT). After washing with D-PBS containing 0.05% Tween-20 (Sigma), serum samples were serially diluted in D-PBS containing 2% bovine serum albumin fraction V and 5 μg/mL Heteroblock (Omega Biologicals) then added to the plates and incubated for 2 h at RT. After washing with D-PBS containing 0.05% Tween-20, 1:2,500 dilutions of goat anti-mouse H+L chain HRP (Jackson Immunoresearch), goat anti-mouse IgM (μ chain) HRP (Jackson Immunoresearch) or goat anti-mouse IgG (Fcγ) HRP (Jackson Immunoresearch) were added to the plates and incubated for 90 min at RT. Plates were washed with D-PBS containing 0.05% Tween-20 then developed for 5 min with SureBlue Reserve TMB substrate (SeraCare). Reactions were stopped using 1 N HCL (Thermo Scientific) and plates were read at 450λ using a Molecular Devices Spectra Max 340 PC running Soft Max Pro 3.1.2.