Pvdf multiscreen hts ip plates

JHMV-specific IgG ASC were measured by ELISPOT assay as previously described (DiSano et al., 2017 (link)). Briefly, 96-well PVDF MultiScreen HTS IP plates (EMD Millipore, Billerica, MA) were coated with JHMV DM (∼5 × 10⁵ PFU/well) overnight at 4 °C. Serial dilutions of cells plated in triplicate were incubated for 4 h at 37 °C and 5% CO₂. ASC was detected by sequential incubation with biotinylated rabbit anti-mouse IgG (0.5 μg/ml; Southern Biotech, Birmingham, AL) overnight at 4 °C, streptavidin horseradish peroxidase (1:1000; BD Biosciences, St. Louis, MO) for 1 h at room temperature, and filtered 3,3′-diaminobenzidine substrate (Sigma-Aldrich, St. Louis, MO) in 0.3% hydrogen peroxide. Brown spots were visible within 2–4 min and the reaction was terminated using cold tap water. Spots were counted using an ImmunoSpot ELISPOT reader (Cellular Technology Ltd., Shaker Heights, OH). Minimum and maximum spot size cutoffs were set to 0.0009 mm² and 0.2295 mm², respectively and spots were analyzed using diffuse processing and spot separation size of 3.00-5.00. Following automated counting, wells were re-counted manually for exclusion of artifacts. Wells containing ≥4 spots scored positive for virus-specific ASC. For analysis, 3–5 wells within a linear dilution range were averaged for each stimulation condition.