CyRPA Binding Assay Protocol

96-well NUNC immunoplates (Thermo Fisher Scientific) were coated with 50 μL of either native state or denatured (boiled at 95 °C for 5 min and reduced using 5 mM dithiothreitol) CyRPA at 2 μg/ml overnight at 4 °C. The plates were then washed with PBS/Tween-20 (PBS-T) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.05 % Tween-20) three times. Next, plates were blocked with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄) with 1% casein for 60 min at room temperature. Plates were then washed with PBS-T before the addition of 50 μL of mAb at 10 μg/ml, diluted in PBS-casein. mAb solution was incubated for 60 min at room temperature before washing with PBS-T. Next, 50 μL of goat anti-human whole IgG alkaline phosphate conjugate (Sigma-Aldrich), diluted 1/1000 in PBS with 1% casein, was added to each well and then incubated for 60 min at room temperature, then washed. Next, a 20 mg tablet of 4-Nitrophenyl phosphate (pNPP, Sigma-Aldrich) was dissolved in 4 ml of diethanolamine buffer (Thermo Fisher Scientific) with 16 ml deionized water. 100 μL pNPP was then added to each well and plates were allowed to develop until the positive control (mAb c12) reached an OD₄₀₅ of approximately 2.0. The anti-RH5 mAb, R5.011^{5 (link)}, was used as a negative control as it should not bind to CyRPA.