ELISA-based Quantification of Protein Binding

Nunc polysorb plates (Thermo Scientific, Germany) were coated overnight with indicated amounts of recombinant E ectodomain protein or E-quadruple mutant (in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃ pH 9.6)) per well with gentle agitation at 4°C. The plates were washed three times with 350 μL per well of PBS/Tween (0.05%), followed by blocking with 5% non-fat dry milk powder (200 μL per well) for 2 h at room temperature (RT). After a second wash step, human sera (dilution 1:100 in 5% non-fat dry milk powder, 100 μL per well) were incubated for 1.5 h at RT. The sera were removed by a third wash step and 100 μL of the secondary antibody (1:10.000 diluted HRP-conjugated Goat-anti-Human IgG (Fisher Scientific)) was added for 1 h at RT. After washing, the TMB-substrate (BioLegend, Germany) was added to the wells and the plate was incubated for 30 min at RT in darkness. To stop the reaction, 1 M H₂SO₄ was added, followed by measurement at 450 nm and 520 nm (reference wavelength) in an ELISA Reader (Infiniti M200, Tecan). All antibody tests were performed in duplicates in at least two independent experiments.
Equal loading of wild type and mutant E protein was verified using the humanized E16 monoclonal antibody (dilution 1:1000), which targets an epitope on domain III of the E protein, distant from the fusion loop [23 (link)] (data not shown).