Hrp conjugated donkey anti mouse igg antibody

To measure virus-specific IgG levels, Thermo Scientific Maxisorp plates (Thermo Fisher Scientific) were coated with 1×10⁵ PFU of heat-inactivated (30 minutes at 56°C) ZIKV^CDN or ZIKV^BR overnight at 4°C. Following incubation, plates were washed with 2x with PBS-0.1% Tween and blocked for 2 hours in 5% milk diluted in PBS-0.1% Tween at room temperature. Serum was collected from naïve or infected mice as described above and diluted in 5% milk PBS-0.1% Tween and incubated for 1 hour at room temperature (serum from infected mice was incubated in wells coated with the matching virus isolate). Wells were washed with 2x with PBS-0.1% Tween. For avidity determination, various concentrations of ammonium thiocyanate (4–0.15M range) were added to each well and incubated for 15 minutes prior to washing 3x with PBS-0.1% Tween. Wells were then incubated with HRP-conjugated Donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, 1:20,000 dilution in 5% milk PBS-Tween solution) for 1 hour at room temperature followed by washing 3x in PBS-0.1% Tween. Plates were reacted with TMB substrate and reaction stopped with 4N Sulfuric Acid prior to being read at 450nM using an AccuSkan FC plate reader.