Diethanolamine

Background correction was performed by subtracting the highest MFI background signal (PBS-TBN, IgG1+IgG3, IgG1, or IgG3 mAb pool binding signals, dependent on the studied IgG subclass) from the sample MFI signal. Heatmaps were generated in GraphPad Prism 7.03.
Nonlinear regression fits were calculated and concentrations at half-maximal binding (EC50) determined (GraphPad Prism). A standard ELISA protocol was followed; the antigens (1 μg/mL) were adsorbed onto ELISA plates (Immulon 4HBX; Thermo Fisher, Waltham, MA) overnight at 4°C. Plates were blocked with 3% BSA diluted in PBS/0.05% Tween-20 for 1 h at room temperature (200 μL per well), to reduce non-specific binding. Plasma samples were analyzed at 1:100 dilutions, IgG samples at 1:5 serial dilutions starting with 500 μg/mL diluted in 0.1% Triton-X, for 1.5 h at 37°C. Alkaline phosphatase-conjugated anti-human IgG (1:2,000; Southern Biotech, Birmingham, AL) was used as a secondary Ab. P-nitrophenyl phosphate tablets (Thermo Fisher, Waltham, MA) dissolved in diethanolamine (Thermo Fisher, Waltham, MA) were used as the substrate. The optical density was read on a microplate reader (Tecan Sunrise) at 405 nm.

For the quantification of reporter gene expression, we utilized a SEAP chemiluminescent assay. The reaction buffer was composed of 5.1 M diethanolamine (Thermo, USA), PNPP substrate tablet (Thermo, USA), L-homoarginine (Alfa Aesar, USA), and 10 mM MgCl₂ (SG-BIO, Republic of Korea). Before adding the reaction buffer, the samples were preheated at 65°C for 1 h to inhibit endogenous alkaline phosphatase activity. Reaction solution (100 μl) and samples (40 μl) were mixed and carefully added to 96-well plates to prevent bubbles and incubated at 37°C for 10 min. After incubation, PNPP (p-nitrophenyl phosphate) solution was added to determine the kinetics using a microplate reader (Infinite F50, TECAN) at 405 nm absorbance in 30 s intervals for 1 h. The Vmax value was calculated using TECAN Magellan software.

ELISA plates were coated with ERA soluble glycoproteins (a.a. 1–439). Plates were washed and then blocked with ELISA blocking buffer (EBB; PBS, 1% BSA, 0.05% Tween). Antibody samples were added to the plate and then diluted 1:2 in EBB. Samples were detected with a goat anti-human IgG conjugated with Alkaline Phosphatase (1:500, Jackson ImmunoResearch). The plates were developed with p-Nitrophenyl phosphate disodium salt at 1 mg/mL in 1 M diethanolamine (ThermoFisher) for 20 min, and then analyzed at 405 nm with a Vmax plate reader (Molecular Devices).