Tmb solution

ELISA plates (Nunc MAXIsorp, from Thermo Fisher Scientific) were coated with 100 ng/well of recombinant NiV sG protein or 50 ng/well of recombinant NiV mcsF protein in 100 μL 0.06 M carbonate/bicarbonate buffer pH 9.6 (Sigma-Aldrich). Plates were blocked with 5% skimmed milk (Marvel) in PBS at 37 °C for 2 h. Porcine serum samples were diluted two-fold starting at 1:400. Horseradish peroxidase conjugated anti-porcine IgG rabbit polyclonal antibodies (Merck, Livingstone, UK) were added at 1:10,000 for 1 h at 37 °C. TMB solution (Merck) was added and plates incubated for 3 min at room temperature in the dark before the addition of 2N sulfuric acid stop solution. Absorbance was read at 450 nm using the GloMax^® Multi+ Detection System (Promega, Chilworth, UK). Antibody end-point titers were calculated as the reciprocal of the highest dilution at which the OD value was greater than the cut-off value (mean + 3 standard deviations of a negative serum).

Albumin (from bovine serum and human serum), glutaraldehyde (25% in H₂O), Tween 20, casein, phosphate buffer saline (PBS), Tris-hydrochloride (Tris–HCl), and TMB solution were purchased from Merck (Darmstadt, Germany). HRP and Bradford Quick Start dye (Coomassie Blue G250) were purchased from Thermo-Fisher Scientific (Waltham, USA). Ethanol (> 99%, extra pure) was purchased from Daejung Chemistry (Siheung, Korea). ProA was purchased from BioVision (Milpitas, USA). Amicon filters (3 kDa cutoff) were purchased from Merck. Transmission Electron Microscopy (TEM) grids (only carbon film, 200 mesh) were purchased from Ted-Pella (Redding, USA). Human Trx1 and IgG1-isotype anti-Trx1 antibody from mouse (ab57675) were purchased from Abcam (Cambridge, UK), and anti-Trx1 rabbit polyclonal antibody (14999-1-AP) was purchased from Proteintech (Rosemont, USA). HRP-conjugated polyclonal antibody from rabbit (MBS715155) was purchased from MyBioSource (USA).

The ssDNA and target DNA were procured from Bioneer. The specific ssDNAs used were [Thiol]-5′-ATATATATATA-3′-[Biotin] and [Thiol]-5′-ATATATATATA-3′. For the targets, the following DNAs were utilized: HPV16L1: 5′-GTGCTGCCATATCTACTTCA-3′, HPV18L1: 5′-TGTGTAGAAGCACATATTGT-3′, HBV: 5′-TTGGCTTTCAGTTATATGGATGATGTGGTA-3′, and BRCA-1: 5′-GAACAAAAGGAAGAAAATCA-3′. The crRNA and Cas12a were obtained from IDT, with the crRNAs used being HPV16L1 crRNA: 5′-UAAUUUCUACUAAGUGUAGAUUGAAGUAGAUAUGGCAGCAC-3′ and HPV18L1 crRNA: 5′-UAAUUUCUACUCUUGUAGAUACAAUAUGUGCUUCUACACA-3′. Terminal transferase, streptavidin-HRP, dATP, and TMB solution were sourced from Sigma-Aldrich. Biotin-16-dUTP was obtained from Jena Bioscience. The sample was characterized using field emission scanning electron microscopy (FE-SEM; Gemini500, Zeiss, Jena, Germany) and Cs_corrected field emission transmission electron microscopy (FE-TEM; JEM-ARM200F, Jeol, Tokyo, Japan); the microscopes were installed in the Center for University-wide Research Facilities (CURF) at Jeonbuk National University. Fluorescence spectra and absorbance spectra were collected using a multi-mode microplate reader (Tecan, TECAN infinite 200 PRO, Männedorf, Switzerland).

Interferon (IFN)-γ tumor necrosis factor (TNF)-α, and interleukin (IL)-2 were measured in the supernatant of cell-cultured medium or the plasma using the ELISA system (BioLegend). Anti-mouse dsDNA IgG antibodies in the plasma were measured using the ELISA system (Wako Pure Chemical Industries). For the measurement of plasma NP-specific IgG antibodies, a MaxiSorp 96-well plate (Nunc) was coated with 10 μg/ml of NP-BSA in PBS at 4°C overnight and blocked with 2% normal goat serum in Tris-buffered saline with 0.05% Tween 20 (TBS-T). Plasma samples diluted 1: 100 were incubated at room temperature for 2 h, and subsequently HRP-conjugated goat anti-mouse IgG was incubated for 2 h. HRP activity was evaluated with TMB solution and 1 M sulfuric acid (Sigma Aldrich). The total urinary protein (uTP) and urinary creatinine (uCr) concentrations were measured by clinical diagnostic reagents (Wako Pure Chemical Industries).

In order to verify whether the vaccine polypeptide can stimulate helper T cell immunity, the ELISA experiment was conducted to determine IFN-γ. In 96-well plates with RPMI1640 medium containing 10% FCS, we co-culture the synthesized vaccine polypeptide (5 μg/ml) with PBMCs (2 × 10⁵) of CE and HC groups. After culturing at 37°C for 20h under 5% CO₂, the ELISA plate was coated with the specific anti-Human IFN-γ capturing antibody in coating buffer overnight at 4-8°C. The plate was blocked with phosphate buffer saline (PBS) containing 5% nonfat milk for at 37°C for 3 h and washed by PBS containing 0.05% Tween 20. Then, the collected cell culture supernatant was added and the plate was incubated at 37°C for 1 h. After washing the ELISA plate with PBS containing 0.05% Tween 20, the specific anti-IFN-γ secondary antibody labelled with HRP was added and incubated at 37°C for 1 h. Last, after washing again and the TMB solution (sigma) was added for color development. The OD_450nm value was measured using a Microplate reader and the levels of IFN-γ was reflected by standard curve. The Human IFN-γ ELISA Kit (70-EK180-48, MultiSciences, Hangzhou, China) was adopted for the ELISA experiment.

Saliva pooled under the tongue was drooled into a 50 mL tube and stored at −80 °C until analyzed. Immuno MaxiSorp 96-well ELISA plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 2 µg/mL recombinant SARS-CoV-2/2019-nCoV S1 protein (Sino Biologicals) diluted in PBS. Wells were blocked with 10% skim milk in PBST (PBS + 0.1% Tween 20) at room temperature for 1 h. Two-fold serial dilutions of saliva samples in PBST were transferred to the ELISA plates (in duplicate) and incubated at room temperature for 1 h. Saliva from an asymptomatic individual confirmed negative for SARS-CoV-2 by clinical testing was used as a negative control. Saliva from a convalescent individual recently infected with SARS-CoV-2 was used as a positive control. Antibody binding was detected with biotinylated anti-human IgA (1:5000; Sigma-Aldrich) and IgG (1:10,000; Assay Matrix) for 1 hour at room temperature, then Streptavidin-HRP (1:5000; Life technologies) in PBST for 45 min at room temperature. Color was developed with TMB solution (Sigma-Aldrich) and H₂O₂ with the reaction stopped using 2 M H₂SO₄. Absorbance at 450 nm was read on a microplate reader. Examples of titrations are shown in Supplementary Fig. 2. OD values with negative control saliva were subtracted from the test samples at each dilution, then endpoint titers calculated.