Tween 20

A chopped glass fiber conjugate pad cut to 29 mm width (Lydall Performance Materials, Rochester, NH, Lypore 9818) and blocked with a solution of 0.075% one-day aged casein (from 1% stock), 5 mM borate (ThermoFisher, Wattham, MA, 28341), 0.05% Tween-20 (Seracare, Milford, MA 5460–0019) and 0.2% w/v sucrose (Sigma, St Louis, MO, S7903). The entire conjugate pad was submerged in the blocking solution for 15 minutes with continuous rocking. The nitrocellulose was removed and dried in a 40°C forced-air oven for 60 minutes.

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.