Costar 3590

The expression of IL‐1β, TNF‐α, IL‐6, CXCL‐15, NE, and MPO was detected by the DuoSet ELISA kit (R&D System, USA). Briefly, the capture antibodies were coated in a 96‐well enzyme‐linked immunoassay plate (Costar 3590, Corning Incorporated, USA) on the first day at room temperature. After being treated with washing buffer, skin lysate and serum were incubated in the plate for 2 h. Then, the detection antibodies were treated, and absorbance at 450 nm was detected by a microplate (Safire, Tecan, Switzerland).

A measure of 50 µL purified antigen (concentration of 4 µg/mL RBD in PBS) was used to coat the 96-well microtiter plates (costar 3590, Corning Incorporated, Kennebunk, ME, USA) over night at 4 °C. Afterwards, the plates were blocked for 1 h at RT with 10% FCS in PBS. Between each step, the plates were washed 3 times with PBS-T (0.05% Tween). The sera were pre-diluted 1:100 in 10% FCS in PBS and incubated for 1.5 h at RT. For detection, anti-human IgG-, IgM- or IgA-HRP-linked ECL Ab (Cytiva, Dassel, Germany) was used in a 1:3000 dilution. Between each step, the plates were washed 3 times with PBS-T (0.05% Tween). The ELISA plates were developed with 75 µL TMB ELISA Substrate Solution (eBioscience, San Diego, CA, USA) for 5 min, stopped with 75 µL 1 N sulfuric acid and analyzed directly at 450 nm on a Tecan reader (Tecan Group, Männedorf, Schweiz).

The IgG titer to KLH-OspA_221–240 and UNC-OspA_221–240 was determined by enzyme linked immunosorbent assays (ELISA). Ninety-six well plates (Costar 3590; Corning) were coated with 1 mg of full-length r-OspA per well in carbonate buffer (pH 9.6; overnight; 4 °C) and non-specific binding blocked (5% non-fat dry milk in PBS with 0.2% Tween-20 (PBSTM); 2 h). Serial dilutions of α-KLH-OspA_221–240, UNC-OspA_221–240 antiserum or α-KLH antiserum (1:50–1:109,350) were added to the wells (1 h; room temperature; in triplicate), the plates were washed 3 times (PBS-T) and HRP-conjugated goat α-mouse IgG (secondary Ab; 1:15,000) was added with ABTS serving as the chromogenic substrate. Absorbance was read at 405 nm in an ELISA plate reader (ELix 808; Biotek) during the linear phase of the reaction. Titers were calculated by fitting a logarithmic curve to the absorbance curve and calculating the inverse dilution corresponding to 1/3 of the maximum absorbance plateau as previously described [27 (link)]. Statistical analyses are detailed in figure legend 2.

ELISPOT assay to detect CSFV-specific IFN-γ cells was performed as previously described [34 (link)], using PBMCs that were obtained at day 0 and at the time of euthanasia. Briefly, plates (Costar 3590, Corning) were coated overnight with 5 μg/ml capture antibody (P2G10, Pharmigen). Detection was performed using a biotinylated antibody (P2C11, Pharmigen). A total of 5x10⁵ PBMCs/well were plated in triplicate at 0.1 multiplicity of infection (MOI) of the Cat01 and Margarita CSFV strains. Moreover, the same samples were incubated in the presence of phytohaemagglutinin (PHA) (10 μg/ml). The controls were incubated in the presence of mock-stimulated wells. The numbers of spots in the media for mock-stimulated wells were considered to constitute the baseline for the calculation of antigen-specific frequencies of IFN-γ-producing cells.

The amount of SARS-CoV-2 spike RBD-specific antibodies was quantified using an in-house ELISA described previously⁷⁴ . Briefly, purified RBD protein was used for coating at a concentration of 5 µg/ml and 50 µl per well in a 96-well microtitre plate (Costar 3590, Corning Incorporated, Kennebunk, USA). For the ChAd-Y25 titre, the same ELISA approach was used, and 5 × 10⁸ viral particles/well of the Vaxzevria vaccine (AstraZeneca, Oxford) were used for coating. After overnight coating at 4 °C, 230 µl of 10% FCS in PBS per well was used for blocking. Blocking was performed for 1 h at RT. Plates were washed four times with PBS-T (PBS containing 0.05% Tween). 50 µl of in blocking buffer 1:100 diluted sera were incubated in the wells for 1.5 h. An HRP-linked anti-human IgG antibody (Cytiva, Cat. NA933-1ML, Dassel, Germany) at a dilution of 1:3000 was used as a secondary antibody and incubated for 1.5 h on the plates. After washing five times, plates were developed for 5 min with 100 µl TMB solution (eBioscience, San Diego, USA) and stopped with the same volume of 1 N sulphuric acid. Absorbance was measured directly at 450 m on an Infinite M1000 reader (Tecan Group, Männedorf, Switzerland).