Elisa plate

ELISA plates (96-well, Thermo Fisher, Waltham, MA, USA) were coated with 100 ng of peptides or recombinant tau proteins in 100 µL phosphate-buffered saline (PBS) for peptides or H₂O for proteins per well. Wells were washed with PBS and blocked with 5% FBS/PBS. Primary antibodies were added to blocking solution and incubated at room temperature. After PBS washes, plates were incubated with goat anti-mouse IgG + IgM (H + L) horseradish peroxidase (HRP)-conjugated antibody (Jackson Immuno Research Labs, West Grove, PA, USA) in 5% FBS/PBS. Plates were washed with PBS and 3,3’,5,5’-tetramethylbenzidine (TMB substrate, Thermo Fisher Scientific, Waltham, MA, USA) was added to each well. The reactions were stopped by adding HCl and the optical density was measured at 450 nm with a plate reader.

Blood samples were obtained between 08:00 and 09:00. Periostin levels were measured by Shino Test Corp. (Kanagawa, Japan) using an enzyme-linked immunosorbent assay (ELISA), as described previously.¹⁸ (link) Briefly, The SS18A mAb (2 μg mL⁻¹) was incubated overnight at 25 °C on ELISA plates (Thermo Fisher Scientific, Rochester, NY, USA). Then the ELISA plates were blocked by blocking buffer (0.5% casein in TBS, pH 8.0) overnight at 4 °C and then washed three times with washing buffer (0.05% Tween20 in PBS). To measure periostin levels, diluted (1/100–1/200) serum samples or recombinant periostin standards were added and incubated for 18 h at 25 °C. After washing five times, the peroxidase labeled SS17B mAb (50 ng mL⁻¹) was added followed by incubation for 90 min at 25 °C. After washing five times to remove excess Ab, reaction solution (0.8 mM 3,3′,5,5′-Tetramethylbenzidine, 2.5 mM H₂O₂) was added, followed by incubation for 10 min at 25 °C and then the reaction was stopped by adding the stop solution (0.7 N HCl). The values were calculated by subtracting the absorbance at 550 from the absorbance at 450 nm. Periostin concentrations in the serum were calculated simultaneously using the recombinant periostin proteins. The assay was performed in duplicate.

WA-1 or Beta SARS-CoV-2 spike-specific IgG titers were assessed with an antibody titration ELISA as described previously [6 (link)]. Briefly, ELISA plates (Thermo Fisher Scientific, 442404) were coated with 1 µg/mL of SARS-CoV-2 WA-1 S-2P or S-2Pᵦ in PBS (pH 7.4) at 4 °C for 16 h. The plates were washed with PBST three times, then blocked with 5% skim milk in 1 × PBST at RT for 2 h. The sera were diluted by 100-fold in 5% skim milk in PBST. A further serial 4-fold dilution for week-0 and week-2 sera or 6-fold dilution for week-6 sera was applied to the 100-fold dilution preparations. The plates were incubated with diluted sera at RT for 1 h. The HRP-conjugated anti-mouse secondary antibody (Thermo Fisher Scientific, 1/2000 dilution) was used to detect the antibody responses. The endpoint titers were calculated as the dilution that yielded an optical density equivalent to 4× background (secondary antibody alone).

Total antigen-specific IgG levels were determined by ELISA. ELISA plates (Thermo Scientific) were coated with full length proteins at 0.05 μg/well in carbonate buffer (pH 9.6) and incubated overnight at 4 °C. After washing with PBS, containing 0.05% Tween 20, the plates were incubated with blocking buffer (10% of fetal calf serum (FCS, Biowest) in PBS) for 1 h. Mouse sera were added at 1:5600 dilution and incubated for 2 h at room temperature. After washing, goat anti-mouse total IgG conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch) was added (1:1000 dilution) in blocking buffer and incubated for 1 h at room temperature. The plates were then extensively washed and incubated with KPL SureBlue substrate. The reaction was stopped with 2N H₂SO₄. Absorbance (450 nm) was immediately measured using a BioTek Synergy HT multi-detection microplate reader.

ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 50 ng/well of purified VLPs and incubated at 37 °C for 2 h, followed by blocking with 5% non-fat milk diluted in PBST. Then, hybridoma culture supernatants (50 μL/well), anti-GII.3-VLP mouse sera (1/1000 dilution), anti-GII.3 mAbs (50 ng/well), a norovirus cross-reactive mAb 7D8 (50 ng/well) [38 (link)], or an irrelevant anti-SARS-CoV-2 mAb 2H2 (50 ng/well) [41 (link)] were added to the plates and incubated for 2 h at 37 °C. After washing, the plates were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 37 °C. After washing, color was developed with the TMB substrate system, and the absorbance was then determined at 450 nm wavelength.