Immunoreactive Profiling of Bacterial Antigens

Formalin-fixed (FF) bacteria (2–4 × 10⁸ CFU total) or outer membrane protein (OMPs) fractions (5 µg total) isolated using Sarkosyl as previously described [34 (link)] were spotted on a nitrocellulose filter using a slot-blot apparatus (Thermo Fisher Scientific). The membranes were blocked with 1% BSAin TBS/Tween 20 (TBS-T) and incubated with pooled sera aliquots (1:200 dilution) overnight at 4 °C. An anti-mouse IgG secondary AP-conjugated antibody (Southern Biotech, Birmingham, AL, USA) was used to detect immunoreactive dots with NBT/BCIP (5-bromo-4-chloro-3-indolyl phosphate/Nitroblue Tetrazolium) chromogenic substrate (Bio-Rad, Hercules, CA, USA).