Horseradish peroxidase conjugated goat anti mouse igg h l

Serum anti-DNP total IgG was assessed using an ELISA. 96-well polysorp plates were coated for 1 h at rt (21°C) with 5 μg mL⁻¹ DNP₈-BSA (DNP-conjugated BSA; Biosearch Technologies) in PBS. Washing steps were performed 3 times with 0.05% Tween-20 in PBS (PBS-T) and the plate was blocked for one hour at RT with 1% BSA in PBS-T (BSA/PBS-T). The plates were washed, and dilutions of serum in BSA/PBS-T were added and incubated for 2 h at rt. Again, the plates were washed, and horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (SouthernBioTech) diluted 4000:1 in BSA/PBS-T was added. Following a 2 h incubation at rt, the plates were washed. Wells were then incubated with 50 μL Ultra TMB (ThermoFisher Scientific) for 5 min then quenched with 50 μL 1 aqueous sulfuric acid. Plates were read at 450 nm. To measure titers for each IgG antiserum, absorbance curves were fitted, and the titer was assigned as the dilution giving an absorbance of 0.3 O.D. This cutoff value was determined as the mean plus ≥3 times the standard deviation of the negative control group (untreated mice). If no dilution gave an absorbance at or above the cutoff, the titer was considered to be 10². Negative control mice did not raise detectable IgG.