High binding 96 well elisa plates

Protein extract of human laryngeal carcinoma cells (ATCC^® CCL‐23^™) was prepared from trypsinized HEp‐2 cell monolayers using hypotonic lysis buffer (20 mM Tris‐HCl, pH 7.5, 5 mM MgCl2, 5 mM CaCl2, 1 mM DTT, 1 mM EDTA, plus proteases inhibitors (Pierce) as previously described 40. High‐binding 96‐well ELISA plates (Costar) were coated overnight with 0.5 μg/well of HEp‐2 whole cell lysate in PBS. After washings with PBST, plates were blocked 2 h with the ELISA blocking buffer. After washings with PBST, coated plates were incubated 2 h with IgG or IgA antibodies diluted at 6.67 nM in PBS (in triplicate), and revealed as described above. Recombinant IgG and IgA antibodies (150 μg/mL), and control antibodies (mGO53 8, ED38 38, and internal controls in the kit) were analyzed by indirect immunofluorescence assay (IFA) on HEp‐2 cells (ANA AeskruSlides, Ingen) sections using FITC‐conjugated anti‐human IgG antibodies as the tracer according to the manufacturer’ instructions. Sections were examined using the fluorescence microscope Axio Imager 2 (Zeiss) with ApoTome.2 system, and pictures were taken at magnification ×40 with 7000 ms‐acquisition using ZEN imaging software (Zen 2.0 blue version, Zeiss) at the Imagopole platform (Institut Pasteur).

Blood was collected from WT and STAT6 KO mice 5 days after immunization and serum was obtained and stored in −20 °C. Specific anti-Ovalbumin antibodies (anti-OVA) were analyzed by ELISA, exactly as previously described (Sulczewski et al., 2020 ). Commercially available purified Ovalbumin (Sigma) at a final concentration of 2 μg/mL was used to coat high binding 96-well ELISA plates (Costar). Sera were serially diluted (dilution factor = 3) starting at 1:100. Anti-mouse IgG conjugated with HRP (SouthernBiotech), anti-mouse IgM, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, and anti-mouse IgG3 conjugated with HRP (all purchased from The Jackson ImmunoResearch Laboratory) were used to detect antigen-bound primary antibodies. Titers represent the highest serum dilution showing an OD₄₉₀ read higher than 0.1. The endpoint titers were normalized on a log10 scale.

Binding of GP to NPC1 domain C was performed as previously described (10 (link), 32 (link)). Briefly, high-binding 96-well ELISA plates (Corning) were coated with the anti-GP monoclonal antibody KZ52 (2 µg/ml in PBS) (21 (link)). Following a blocking step, either uncleaved or in vitro-cleaved GP_CL pseudotypes were captured on the plate. Unbound GP was washed off, and serial dilutions of Flag-tagged purified soluble human NPC1 domain C (0 to 40 µg/ml) were added. Bound NPC1 domain C was detected by a horseradish-conjugated anti-Flag antibody (Sigma-Aldrich), using ultra-TMB substrate (Thermo Scientific). EC₅₀s were calculated from binding curves generated by nonlinear regression analysis using GraphPad Prism software. Binding ELISAs were done in duplicate in at least two independent experiments. All incubation steps were done at 37°C for 1 h or at 4°C overnight.