Biotinylated goat anti human igg and hrp conjugated streptavidin

The protocol for detection of plasma IgG against intact whole-cell bacteria was performed as previously reported (29 (link), 36 (link)). Individual wells of 96-well polystyrene plates were coated with 10⁶ CFU of PFA killed bacteria in carbonate-bicarbonate buffer pH 9.6 overnight. After washing with 0.1% Tween 20 in PBS, the plate was blocked nonspecific binding with 10% fetal bovine serum (FBS) in PBS for 2 h at room temperature. Heparinized plasma was diluted 1:50 in 0.05% Tween 20, 10% FBS in PBS before adding to the plate, and incubating for 2 h at room temperature. The plate was washed and added for biotinylated goat anti-human IgG and HRP conjugated streptavidin (BD Biosciences) and then incubated 1 h at room temperature. After washing, color was developed for 15 min by using TMB Substrate Reagent Set (BD Biosciences). The reaction was then stopped by adding 2 N H₂SO₄. Absorbances were measured at 450 nm. The results were analyzed and shown as arbitrary units/mL (AU/mL) by comparing it with absorbances from an in-house prepared human IgG standard curve.

Bacterial lysates of clinical Acinetobacter species (500 μg) were separated by 12% to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then blotted onto a PVDF membrane with a wet electrophoresis system (Bio-Rad, Hercules, CA, USA), and the membrane was blocked with 5% skimmed milk for 1 h at room temperature. Heparinized plasma was diluted to 1:100 with 0.1% TBST, then added onto the membrane and incubated overnight. The membrane was washed with 0.1% TBST and biotinylated goat anti-human IgG and HRP conjugated streptavidin (BD Biosciences) was added for 1 h at room temperature. The membrane was washed and SuperSignal West Femto (Thermo Fisher Scientific) was added for signal detection, captured using a ChemiDoc XRS imaging system (Bio-Rad) and analyzed with Quantity One (Bio-Rad) software. Densitometry of individual bands was performed using the GelAnalyzer software. Coomassie blue staining of bacterial lysates and IgG binding on Western blot are shown in Fig. S2.