Elispot aec substrate set

Mouse splenocytes were used to enumerate NP-specific antibody secreting cells 21 days post-vaccination. ELISPOT plates (Millipore Sigma cat. no. S2EM004M99) were coated with 10μg/ml recombinant NP (Sino Biological inc, cat. no 11675-V08B) in PBS and then incubated overnight at 4°C. Mouse splenocytes were processed as outlined above and then 3x10⁶ splenocytes in 100μl of media were plated in NP-coated plates in duplicate and then incubated overnight (~16hrs) at 37°C. The following day, the cells were discarded and the wells were washed 5 times with PBS. Following the final wash, 100μl of anti-mouse IgG-HRP, IgG₁-HRP, or IgG_2C–HRP diluted at 1:2000 in PBS were added to each well. Spots were developed using BD ELISPOT AEC substrate set (BD cat. no. 551951) and then visualized using a CTL imager (Cellular Technologies) and were enumerated using an ImageJ cell counter.

SARS-CoV-2 S1 and S2 protein-specific IFN-γ releases were measured using the method of enzyme-linked immunosorbent spot (ELISPOT) assays (Cat# 551083, BD Bioscience, USA) according to a previously described procedure.^{62 (link)} Briefly, the 96-well ELISPOT plates were coated with purified anti-mouse IFN-γ monoclonal antibody overnight at 4°C. Then, the plates were blocked and 2 × 10⁵ fresh splenocytes were added into each well and incubated with peptide pools of S1 or S2 for 20 hours at 37°C in a humidified incubator with 5% CO₂. The final concentration for each peptide was 1 μg/ml. After incubation, detecting antibody and Avidin-HRP were added sequentially. Finally, the plates were developed using the BD™ ELISPOT AEC Substrate Set (Cat#551951, BD Bioscience, USA) according to the manufacturer’s manual. Spots representing IFN-γ producing cells were enumerated using an automated ELISPOT plate reader (ChampSpot III Elispot Reader, Saizhi, Beijing, China). At the same time, the supernatants in the wells of ELISPOT plates were also collected for detecting secreted cytokines using a multiplexed cytokine beads array kit (Cat#741054, Biolegend, USA). The concentrations of secreted cytokines were detected using a BD Fortessa flow cytometer (BD Biosciences, USA). Data were analyzed using the LEGENDplex Data Analysis software suit (Biolegend, USA).

Spleens were harvested and processed individually into single-cell suspensions. 1 × 10⁶ splenocytes were plated onto 96-well plates previously coated with an IFNγ capture antibody (BD Cat# 551083). C57BL/6-CEA Tg splenocytes were stimulated with one of the following  H2-D^b- or H2-K^b-restricted peptides (10 µg/mL) for 18 hours: CEA_526-533 (EAQNTTYL), CEA_572-579 (GIQNSVSA), p15E (KSPWFTTL), and HIV-gag (SQVTNPANI). The CEA_526-533, CEA_572-579, p15E, and HIV-gag peptides were synthesized by CPC Scientific (Sunnyvale, California, USA). IFNγ spots were detected using the BD mouse IFNγ ELISPOT kit and developed using the BD ELISPOT AEC substrate set according to the manufacturer’s instructions (BD Biosciences; San Jose, California, USA). IFNγ spots were visualized and quantified using the CTL ImmunoSpot Analyzer (Cleveland, Ohio, USA).

IFNγ ELISpot assays (BD ELISPOT Mouse IFNγ ELISPOT Set, BD Biosciences) were performed according to the manufacturer’s instructions. Briefly, 96-well ELISpot plates were coated with an IFNγ capture antibody (BD Biosciences, 51-2525K) in PBS overnight at 4 °C. Plates were then blocked with complete growth medium for 2 h at room temperature. Wells were then emptied, and 1 × 10⁶ splenocytes from immunized mice were added to the plates. Cells were arrayed in the presence or absence of 15-mer peptides with 11-residue overlaps derived from the SARS-CoV-2 Spike_RBD (10 μg/mL) in 200 μL of complete medium and incubated overnight at 37 °C. Plates were then washed and incubated with a biotinylated IFNγ detection antibody (BD Biosciences, 51-1818KZ) for 2 h at room temperature and incubated with streptavidin-horseradish peroxidase (BD Biosciences) for 1 h at room temperature. Plates were developed with 3-amino-9-ethyl-carbazole substrate (BD ELISPOT AEC Substrate Set) for 5 min to 30 min and dried overnight. Spots were enumerated using the KS ELISpot analysis system (Immunospot).