Quantification of Plasma IgG against Bacteria

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.