High binding microplate

100 μL of GM1-HSA (0.1 μg mL⁻¹, IsoSep) or tri-LeX-APE-HSA (10 μg mL⁻¹, IsoSep) in PBS were added to high binding microplates (Greiner) and left to incubate overnight at 4 °C for immobilization. Plates were then washed with 0.2% (w/v) BSA (Sigma-Aldrich) in PBS at 37 °C. The plates were then incubated with titrations of each CTB mutant in PBS + 0.2% BSA + 0.05% Tween20 at room temperature for 1 h, followed by rabbit anti-CTB primary antibody (Sigma Aldrich) incubation for 90 mins at room temperature. Anti-rabbit-IgG-HRP secondary antibody (Jackson ImmunoResearch) was then added and incubated for 2 h. Finally, 1-Step Ultra TMB-ELISA substrate solution (Thermo Scientific) was added and followed by addition of sulfuric acid (Sigma Aldrich) to detect the overall binding. The plates were read using ELx800 (BioTek) at 450 nm. The binding of non-biotinylated and biotinylated CTB mutants to GM1-HSA and tri-LeX-APE-HSA were comparable.

For the detection of HA, high binding microplates (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated overnight at 4 °C with 50 µL brain homogenates (5 mg/mL) 1:4000 diluted in carbonate buffer (0.1 M NaCO₃, pH 9.7). Coated plates were washed with TBS-T (0.05% Tween 20) and incubated with blocking solution (1% BSA in TBS-T) for 1 h and probed with biotinylated hyaluronic acid binding protein (Merck Millipore, Darmstadt, Germany, RRID: AB_2861303) at a concentration of 0.125 µg/mL for 1 h. After washing, the plates were incubated with ExtrAvidin^®-Peroxidase (Sigma-Aldrich, Taufkirchen, Germany) for 45 min and developed by using 100 µL/well 3,3′,5,5′-tetramethylbenzidine solution. The reaction was stopped after 25 min with 1 N H₂SO₄ and absorbance was measured at 450 nm (microplate reader Mithras LB 940, Berthold Technologies, Bad Wildbad, Germany).

Total anti-FVIII antibody titers in the blood serum of rhFVIII-treated mice were measured by ELISA. High-binding microplates (Greiner Bio-One) were coated with 1.25 μg/mL rhFVIII diluted in coating buffer (50 mM NaHCO₃ in PBS, Carl Roth) at 4°C overnight. Nonspecific binding sites were blocked for 1 hour at room temperature using 1% bovine serum albumin (BSA, Merck) in PBS. Starting with a 1:20 dilution, plates were subsequently incubated with a serial dilution of serum samples at 4°C overnight. To detect FVIII-specific IgG antibodies bound to the immobilized rhFVIII protein, the plates were incubated with Biotin-SP–conjugated AffiniPure Goat Anti–Mouse IgG (Jackson Immuno Research) at a 1:10,000 dilution for 2 hours at room temperature. Subsequently, the plates were incubated with streptavidin-labeled horseradish peroxidase (Natutec) at a dilution of 1:5,000 for 1 hour at room temperature. σ-Phenylene diamine dihydrochloride (Thermo Fisher Scientific) and H₂O₂ (Carl Roth) were used as substrates to detect immobilized anti-FVIII antibodies. The reaction was stopped using 1 M H₂SO₄ (Carl Roth).
Antibody titers were expressed as the highest dilution of plasma samples showing a positive result (optical density > 0.2) in the ELISA assay. The starting dilution was 1:20; if this was not sufficient to detect a signal, the dilution was further decreased.

ELISA was performed at room temperature with all sera in duplicate using high-binding microplates (Greiner, Monroe, USA). Briefly, wells were coated with 100 μl of 2 ng/μl purified HBRV Su in PBS for 18 hours and blocked with 1% BSA in PBS for 3 hours. Serum was incubated at 100 μl/well at a 1 : 400 dilution in PBS with 1% BSA (Sigma) for 1 hour. A serial dilution of polyclonal anti-MMTV Env was included on each plate as a standard and then incubated with 100 μl/well donkey anti-human and donkey anti-goat secondary antibodies (Jackson Immuno-Research Lab) for 1 hour. The plate was washed 3 × 5 min after each step using PBS with 0.5% Tween. Plates were developed with 100 μl/well tetramethylbenzidine substrate (TMB, Sigma) for 20 min and then stopped with 50 μl/well 2N H₂SO₄. The absorbance at 450 nm and 540 nm (background) was measured with EMAX Plus Microplate Reader (Molecular Devices, USA) and the cutoff level was established using the reactivity of control samples by adding the mean background level to 3 × S.D. Two-tailed Fisher's exact test was used to assess significant differences in frequency between different groups, followed by calculation of the odds ratio (Baptista–Pike methodology) along with sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratio (Wilson Brown methodology) using Prism 8 software.

Three days before fusion, the mouse was immunized with a final boost (25 µg GST-NOX5 in IFA). After harvesting the mouse splenocytes, the cells were used for fusion with SP2/0-Ag14 mouse myeloma cells (for a detailed description of the process see^{33 (link)}. The hybridoma supernatants were tested by ELISA. Briefly, high-binding microplates (Greiner bio-one) were coated with NOX5-His₆ solution in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) for 2 h at room temperature and were washed with 0.1 M PBS-Tween (0.05% Tween20, pH 7.4, 5x). Concentrated supernatant samples were added to the wells and incubated for 1 h at room temperature. After washing (PBS-Tween, 5x), horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was used (in 1:5000 dilution, Southern Biotechnology) as the secondary antibody. After washing 5 × with PBS-Tween, 3,3′,5,5′—tetramethylbenzidine (TMB) was used as the substrate for peroxidase activity detection, and optical density at 450 nm was measured using Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific). Clones that appeared positive were further evaluated, and the NOX5 reactivity was confirmed by independent methods (Western blot, immunostainings) as well. A similar protocol was used for testing sera from mice, but the samples were serially diluted (5 × serial dilution from 100 × diluted sample) in the second step.