O phenylenediamine dihydrochloride

Diluted recombinant S protein (2 μg/mL) (#RP012383LQ, ABclonal) with PBS was incubated to immobilize on the maxisorp plate (464718, Thermo). After blocking with 1% BSA/PBS, 1 μg of K-874A diluted in 1%BSA/PBS was incubated to bind to S protein. Then, serial diluted recombinant ACE2 (from 1 to 0.06 μg/ml) (ab151852, abcam), subsequent rabbit anti-ACE2 antibody (HPA000288, Atlas Antibodies) and HRP-conjugated anti-rabbit IgG (7074S, Cell signaling) were incubated. To detect K-874A binding to immobilized S protein, serial diluted K-874A (1 to 0.002 μg/ml) in 1% BSA/PBS was incubated, and K-874A which bound to S protein was detected by HRP-conjugated anti-VHH antibody (#128-035-232, Jackson Immuno Research). Between each incubation, wells were washed with PBST. To color, o-phenylenediamine dihydrochloride (p6662, Merck) was used as a substrate of HRP and reaction was stopped with 2 M H₂SO₄. Signaling was quantified by Ensight (Perkin Elmer).

An anti-CV ELISA was developed using an anti-citrullinated protein antibody (rabbit polyclonal, Abcam, Waltham, MA, USA: capture Ab) and anti-vimentin mAb (clone: 3CB2, mouse IgM, Iowa Hybridoma Bank, Iowa City, IA, USA: detection Ab). As a capture antibody, 2 μg/mL of an anti-citrullinated protein antibody was added to each well of a High Binding 96-well plate (Corning, Corning, NY, USA) at room temperature (RT) overnight. After blocking with 1% BSA in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) at RT for 30 min, the wells were incubated with CV, citrullinated BSA or gingival crevicular fluid (GCF) samples at RT for 1 h. As a detection antibody, 2 μg/mL of anti-vimentin mAb was added to each well and incubated at RT for 1 h. After washing with PBS-T, 1:2000 diluted horseradish peroxidase (HRP)-conjugated anti-mouse IgM antibody or HRP-conjugated anti-mouse IgG antibody (Jackson Laboratory, Bar Harbor, ME, USA) was applied to the respective wells and incubated at RT for 1 h. The color development of the substrate, o-Phenylenediamine dihydrochloride (Sigma-Aldrich), reacted to the peroxidase and was measured at a 450 nm spectrum using a microplate reader (Synergy H, BioTek, Santa Clara, CA, USA).

As an antigen, we used recombinant proteins derived from the SARS-CoV-2 (S1 domain, RBD domain; Receptor Binding Domain, N, M, and E), and purification was performed as previously described [18 (link)]. Microtiter plates (Sigma-Aldrich, St. Louis, MO, USA) were coated with 100 µL/well of each antigen (S1 domain, RBD domain, N, M, and E) to a final concentration of 0.1 µg/mL in a coating buffer (50 mM Na₂CO₃/NaHCO₃, pH 9.6). The plates were incubated for one hour at 37 °C and then blocked for 40 min at 37 °C with 200 µL/well of 5% skimmed milk diluted in phosphate-buffered saline (PBS)-Tween 20 (0.05%). For the primary antibody, 100 µL/well of serum samples (1:50 diluted in PBS) or breast milk (direct) by duplicate were incubated for one hour at 37 °C. Finally, plates were incubated with 100 µL/well of monoclonal anti-human IgA (Sigma-Aldrich; 1:500 dilution) and IgG (Sigma-Aldrich; 1:1500 dilution, St. Louis, MO, USA) coupled to horseradish peroxidase (HRP) for one hour at 37 °C. After every step, the plates were washed thrice with 200 μL/well of PBS-Tween 20 0.05% for 5 min. The enzymatic reaction was developed using o-phenylenediamine dihydrochloride (Sigma-Aldrich) and stopped using 2 N H₂SO₄. The optical density (OD) was measured at 492 nm using a microplate reader. Samples with OD > 0.300 were considered positive for both antibodies.

To confirm the presence and accessibility of the epitopes on the capsid surface, viral particles were coated on 96-well plates (Nunc, Roskilde, Denmark). Viruses were inactivated by incubation at 56°C for 30 min, followed by addition of 0.1% SDS. Spectrophotometric measurements at 215 and 225 nm allowed to determine viral protein concentrations, and subsequently 100 ng was coated on 96-well plates. To analyze epitope presence and accessibility on virions, non-denaturated viruses (100 ng) were coated on the plates. After overnight incubation at 4°C, non-specific sites were blocked with 5% milk PBS-Tween for 2 h, then plates were washed and incubated for 1 h with an anti-ovalbumin rabbit polyclonal antibody (AB1225, Millipore, MA, USA). Upon washing, an anti-rabbit IgG peroxidase-linked Ab (NA934, Amersham Biosciences, Saclay, France) was added for 1 h and peroxidase activity was revealed by incubation with the substrate O-phenylenediamine dihydrochloride (Sigma-Aldrich, Lyon, France) for 30 min. The reaction was stopped by addition of 3 N HCl and spectrophotometric readings were performed at 490 nm. Each virus was assayed in sexdecaplicates and the experiments were repeated at least twice.

The levels of IL-4 (sensitivity: 2 pg/mL) and IL-5 (sensitivity: 7 pg/mL) in the BALF were measured using sandwich ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer’s protocols. ELISA was also used to determine the levels of OVA-specific IgE in the serum. Briefly, 96-well microtiter plates were coated overnight with 10 μg/mL OVA in PBS-Tween 20. The plate was washed and blocked, and then the samples were added and the plate was incubated for 2 h. After another washing step, HRP-conjugated goat anti-mouse IgE antibody was added, the plates were washed four times, and 200 μL of o-phenylene diamine dihydrochloride (Sigma-Aldrich) was added to each well. The absorbance was measured at 450 nm after incubation for 10 min in the dark. ELISA was performed three times.

Hemojuvelin surface expression was quantified as described previously 13 (link). In brief, 10⁴ HeLa cells were seeded in 48-well plates and transfected with 0.4 μg of plasmid DNA with 1 μl of Lipofectamine 2000 (Invitrogen). After 12 hrs, the medium was replaced with DMEM supplemented with 2% FBS. After 36 hrs from transfection, cells were fixed with 4% paraformaldehyde. Cells were permeabilized with Triton X-100 in PBS to assess whole HJV expression. Cells were blocked with 5% non-fat milk in PBS, incubated with rabbit anti-HJV (1:1000) and then with the secondary HRP antibody. Peroxidase activity was measured with a HRP substrate (o-phenylenediamine dihydrochloride; Sigma-Aldrich) according to the manufacturer's instructions. The amount of m-HJV was calculated as the ratio between the absorbance of unpermeabilized and permeabilized cells. Background absorbance was subtracted for each sample. Two-tail Student's t-test was used for statistical analysis.