Nunc maxisorp microtiter plates

Nunc Maxisorp microtiter plates (Thermo Fisher Scientific) were coated with 0.65 µg/mL solution of PT, or a 1 µg/mL solution of either FHA, PRN, or Fim2/3 (Reagent Proteins and List Biological Labs) in carbonate buffer and incubated overnight at 4 °C. Plates were blocked with PBS with 0.1% Tween 20 (PBST) including 1% bovine serum albumin (BSA) for 90 min at room temperature. Sera were diluted initially at 1:200 and then serial dilution at 1:2 in PBST with 1% BSA. Sera were incubated on coated plates for 90 min at room temperature. Plates were washed six times with PBST. Secondary antibody was goat anti-human IgG labeled with horseradish peroxidase (Millipore AP113P) at 1:5000 in PBST with 1% BSA. 100 µL of OPD (Sigma) in 50 nM citrate buffer (pH 5.0) was used as enzyme substrate. The color reaction was terminated with 1 M hydrochloric acid. Optical density (OD) was measured at 490 nm. Total IgG antibody titers in international units (IU) were analyzed compared to the WHO serum reference standard (NIBSC; 06/140) for PT, FHA, and PRN. Fim2/3 titers were measured relative to the Fim2/3 IgG titer of the WHO standard in relative units (RU).