Ascent version 2

BALF and fecal homogenate supernatants were used to assess the mucosal IgA concentration using a rat IgA ELISA Quantification Set (A110-102) following the manufacturer’s instructions (Bethyl Laboratories, Montgomery, TX, USA) as carried out in previous studies [39 (link)]. All samples were tested in duplicates. Absorbance was measured using a microplate photometer (Labsystems Multiskan, Helsinki, Finland) and data were interpolated using ASCENT version 2.6 software (Thermo Fisher Scientific, S.L.U, Barcelona, Spain) into standard curves. Results were expressed as fold change considering the mean value of the asthmatic group as 100%.

Anti-RV antibody (Ab) levels in serum, GW, and supernatants from the spleen and MLN cell culture were quantified by ELISA, as previously described [27 (link)]. Briefly, 96-well plates (Nunc MaxiSorp, Wiesbaden, Germany) coated with UV-inactivated 10⁵/mL SA11 particles and blocked with phosphate-buffered saline (PBS) and 1% bovine serum albumin (BSA, 1 h, room temperature (RT)) were incubated with dilutions of sera or GW samples during 3 h at RT. After washing, polyclonal anti-rat Ig conjugated to peroxidase (Dako, Barcelona, Spain) and the substrate was added. The standard used was a pooled serum from the dams of inoculated litters. The absorbance was measured using a microplate photometer (LabSystem Multiskan, LabX) and analyzed with the ASCENT version 2.6 software (Thermo Fisher Scientific).
An ELISPOT technique was used to quantify anti-RV Ig-secreting cells (SC) from spleen and MLN, as in previous approaches [27 (link)], using 96-well nitrocellulose plates (Merck Millipore) coated with 10⁵ particles/mL of viral SA11 or EDIM particles. Each spot obtained corresponded to one anti-RV Ig-SC. Spots were automatically counted by the ELISPOT reader system (AID, Strasberg, Germany) and results are expressed as Ig-SC/10⁶ cells.

TNF-α were quantified in the 24 h supernatants by stimulated splenocytes using Opt-EIA-set (BD Biosciences), as in previous studies [46 (link)]. IgG were quantified in 96 h supernatants by nonstimulated splenocytes with an enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions (BD Biosciences), as previously described [46 (link)]. In both cases, absorbance was measured in a microplate photometer (LabSystems Multiskan) and analyzed using ASCENT version 2.6 software (Thermo Fisher Scientific, Waltham, MA, USA). TNF-α and IgG results are shown as percentage with respect to the control condition (without cocoa), which was considered as 100%.