Flat bottom 96 well microplate

After a checkerboard titration, 100 µl of each rEgAgB (1.5 µg/ml suspended in 100 mM carbonate-bicarbonate buffer, pH 9.6) was used to coat the wells of a flat-bottom 96-well microplate (Greiner Bio-One, Kremsmuenster, Austria) overnight at 4 °C. One hundred microliters of serum samples diluted 1:100 in phosphate buffered saline containing 0.05% Tween 20 (PBS/T) were incubated at 37 °C for 2 h, after which 100 µl of horseradish peroxidase-conjugated anti-human IgG antibody (1:4000 dilution in PBS/T; MP Biochemicals, Santa Ana, CA, USA) was further incubated at 37 °C for 2 h. Color reactions were developed with 100 µl of 1% o-phenylenediamine (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.03% H₂O₂ for 20 min in the dark. Reactions were halted by adding 50 µl of 2 N H₂SO₄ (Sigma-Aldrich). The absorbance was measured at 450 nm on an NEO microplate reader (Biotek, Winooski, VT, USA). Sera from healthy donors and PBS/T were used as negative and blank controls, respectively. All results were determined after correction with appropriate blank. Each sample was independently assayed in triplicate.

After a checkerboard titration, 100 μl of PwAWE and rPwYF (2.5 μg/ml each) suspended in 100 mM carbonate-bicarbonate buffer (pH 9.6) were coated to the wells of a flat-bottom 96-well microplate (Greiner Bio-One, Kremsmünster Austria) overnight at 4°C. Sera diluted to 1:200 in PBS containing 0.05% Tween 20 (PBS/T) or undiluted CSFs (100 μl each) were incubated for 2 h, subsequently with 1:2,000 diluted horseradish peroxidase (HRP)-conjugated anti-human IgG antibody (100 μl; heavy- and light-chain specific, MP Biochemicals, Santa Ana, CA, USA) for 2 h. The color reaction was developed with 1% o-phenylenediamine (100 μl; Sigma-Aldrich) supplemented with 0.03% H₂O₂ for 20 min in the dark. The reaction was stopped using 2 N H₂SO₄ (50 μl; Sigma-Aldrich). The absorbance (abs.) was measured at 450 nm on a NEO microplate reader (Biotek, Winooski, VT, USA). All results were determined after correction with PBS/T blank.