3 amino 9 ethylcarbazole substrate

ELISPOT assays were used to determine H7HA-specific IgG and IgA ASCs. Splenocytes and CLNs were collected from immunized mice 3 weeks after the final vaccination. Multiscreen 96-well filtration plates (Merck Millipore, Darmstadt, Germany) were coated with rH7 proteins (1 µg per well) and incubated overnight at 4 °C followed by blocking with 200 µL/well of complete RPMI 1640 (10% fetal bovine serum [FBS], 1 × penicillin/streptomycin, 1 × sodium pyruvate, 1 × nonessential amino acids, and 100 µM β-mercaptoethanol) at RT for 1 h. Next, 5 × 10⁵ splenocytes or CLN cells were added to each well and incubated at 37 °C for 48 h. After three washes with PBST, HRP-conjugated anti-mouse IgG or HRP-conjugated anti-mouse IgA was added to each well for IgG- or IgA-ASCs, respectively. After overnight incubation at RT, and PBST and two PBS washes, 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich, St. Louis, MO, USA) was added, followed by incubation at RT for 0.5–1 h before washing with double-distilled water. Immunospots were determined using an ELISPOT plate reader (CTL, Inc., Cleveland, OH, USA).

ELISPOT was performed to enumerate Dsg3-specific plasmablasts present in PBMC samples. 96-well ELISPOT assay filter plates (Millipore) were coated overnight with recombinant Dsg3 antigen (3 μg/mL, provided by Hertl Lab) or polyvalent goat anti-human Ig (50 μg/mL, Rockland) in PBS/ 1mM CaCl₂. Plates were washed and blocked by incubation with R10 at 37°C for 2 h. Freshly isolated PBMCs were added to the plates in a dilution series starting at 5×10⁵ cells and incubated overnight at 37°C. Plates were washed with PBS, followed by PBS/0.05% Tween, and then incubated with biotinylated anti-human IgG, IgA, or IgM antibody (Invitrogen) at room temperature for 90 min. After washing, plates were incubated with avidin D-horseradish peroxidase conjugate (Vector laboratories) and developed using 3-amino-9-ethyl-carbazole substrate (Sigma). Plates were scanned and analyzed using an automated ELISPOT counter (CTL, Cellular Technologies).

According to our previous study (35 (link)), Enzyme-linked immunospot (ELISPOT) was used to detect EV-A71-specific IgG- and IgA-secreting cells. We coated 96-well microplates (Millipore, United States) with purified EV-A71 virions (10 μg/ml) overnight and blocked the wells with 3% BSA in PBS. Then, spleen cells (5 × 10⁵) in RPMI-10% FBS were added and incubated overnight at 37°C. Following incubation, the plate was washed, and HRP-conjugated goat anti-mouse IgGs (1:000, Bethyl Laboratories, Inc.) or IgAs (1:000, Bethyl Laboratories, Inc.) was added. After incubating plates for 2 h, the HRP conjugate was removed and washed. Then, 100 μl of 3-amino-9-ethylcarbazole substrate (Sigma) was added and spots were allowed to develop in the dark at room temperature (RT) for 10 min. After ELISPOT analysis was completed, the plate was analyzed using an ImmunoSpot^® S6 UV Reader (Cellular Technology Limited, Cleveland, OH, United States). ImmunoSpot^® v.6.0 Software was used to automatically count the spots for each EV-A71 antigen stimulation condition and medium negative controls. Spots from a total of three wells with 1.5 × 10⁶ spleen cells were determined and considered as one set of data.

IFN-γ secreting cells were detected via ELISpot as previously described [6 (link), 20 (link)]. MultiScreen assay plates (Millipore) were treated with 15 μL of 35% ethanol and washed three times with sterile PBS (Gibco). Plates were coated with 50 μL of 10 μg/mL α-mouse IFN-γ (AN18, Mabtech) and incubated overnight at 4 °C. Plates were washed as before, wells blocked with 200 μL of CTL medium for a minimum of 2 h at 37 °C, 2.5 × 10⁵ lymphocytes were added into wells with or without 10 ng/mL (final concentration) of AT-2 inactivated HIV-1 IIIB viral lysate or 10 μg/mL synthetic 17-mer MPER peptide, GNEQELLELDKWASLWN in CTL medium; additional wells included 50 ng/mL phorbol-myristate-acetate and 300 ng/mL ionomycin as a positive control [33 (link)]. Lymphocytes were incubated over two nights at 37 °C in 5% CO₂, then washed 6 times with PBST, and 100 μL filtered biotinylated IFN-γ detection antibody (Mabtech) was added at 1 μg/mL in 0.5% FBS/PBS for 3.5 h at RT. Plates were washed 6 times with PBST, and streptavidin-HRP (1 μg/ml, Pierce Thermo Fisher Scientific) in 5% FBS/PBS was added for 1 h at RT, followed by 3 PBST and 3 PBS washes. Plates were developed with 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich) for 10 min at RT. Spots were counted in an ImmunoSpot analyzer (Cellular Technology Limited).