Tmb substrate

Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).
Total IgM was quantified by sandwich ELISA. Briefly, 96-well plates (Nunc Maxisorp) were coated with 5 μg/ml goat anti-mouse Ig overnight at 4 °C. The plate was blocked with 2% BSA in Tris-buffered saline pH 7.4, 0.05% Tween-20 (TBS-T), for 1 h at RT. After washing with TBS-T, HRP-labeled goat anti-mouse IgM was added and left to interact for 2 h at RT. After a last TBS-T washing, TMB substrate (Biolegend) was added and color development was stopped with 2 N H₂SO₄. Optical density at 450 and 570 nm was recorded (Synergy 2, BioTek). Optical density values from samples obtained at 570 nm were subtracted from those at 450 nm and background values from blank wells were subtracted from samples.

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).

Half-area 96-well medium binding microtitre plates (Greiner bio-1) were coated overnight at 4°C with 0.5 μg TE/well, followed by blocking with blocking buffer for 2 hours at RT. To investigate the potential inhibition of antibody binding to α-Gal, sera from 24 patients with AGS and 9 healthy controls were preincubated with α1,3-galactobiose (Galα1-3Gal, G203, Dextra Laboratories) at a concentration of 500 μg/mL or with blocking buffer for 2 hours at RT prior to addition to the plate (final dilution of sera was 1:5 for IgE and 1:50 for IgG1). IgE binding was detected using mouse-anti-human-IgE antibody conjugated with HRP (1 hour at RT, 1:2,500; ab99806, Abcam) and IgG1 binding with mouse-anti-human-IgG1 (1 hour at RT, 1:1,000; 555868, BD Biosciences), followed by incubation with HRP-conjugated sheep-anti-mouse-IgG (1 hour at RT, 1:5,000; 515-035-071, Jackson Immunoresearch Laboratories). Binding was visualized by incubation with TMB substrate (BioLegend) for 15 minutes before stopping the reaction with 1 M sulfuric acid. The absorbance was measured at 450 nm. Inhibition of IgE and IgG1 binding was calculated as [100–(OD_450nm inhibitor × 100) /OD_450nm no inhibitor]. The results are expressed as percent inhibition of IgE or IgG1 binding of duplicate determinations with a deviation of under 5 %.