LdpA-specific Igs

The 96-well plates (Nunc MaxiSorp; Thermo Fisher Scientific, Waltham, MA, USA) were coated with rLdpA (0.2 μg/mL in 0.05 M sodium carbonate buffer, pH 9.6) and incubated overnight at 4°C. The rLdpA protein in each well was then blocked with phosphate-buffered saline (PBS) (0.2 g/L potassium chloride, 0.2 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride, 1.15 g/L disodium hydrogen phosphate, pH 7.4) containing 2% BSA. Dilutions of human serum samples (1:200 and 1:10) were prepared for IgG and IgE, respectively. Dilutions of mouse serum samples (1:65,536, 1:512, and 1: 10) were prepared for IgG1, IgG2a, and IgE, respectively. The LdpA-specific absorbance values were determined with biotinylated anti-human IgG monoclonal antibody (mAb) (G18-145; BD Biosciences), biotinylated anti-human IgE mAb (f0822; Biomatrix Research, Chiba, Japan), biotinylated anti-mouse IgG1 mAb (A85-1; BD Pharmingen, San Diego, CA, USA), biotinylated anti-mouse IgG2a mAb (R19-15; BD Pharmingen), and biotinylated anti-mouse IgE mAb (R35-72; BD Pharmingen). Biotinylated mAbs were detected with Pierce High Sensitivity Streptavidin-Horseradish peroxidase (HRP) (Thermo Fisher Scientific, Waltham, MA, USA) and tetramethylbenzidine substrate (Bio-Rad, Hercules, CA, USA). The reaction was quenched using 1 N sulfuric acid, and absorbance was measured at 490 nm (A₄₉₀) using an automated plate reader (Sunrise Rainbow Thermo RC; Tekan, Männedorf, Switzerland).