Elispot plate

The instructions for ELISpot Flex: Mouse IgG (ALP) (Mabtech, Nacka Strand, Sweden) were followed. Briefly, PVDF membranes in the wells of ELISpot plates (Merck Millipore, Billerica, MA, USA) were first activated with 20 μL of sterile 35% (v/v) ethanol solution on the first night, followed by five washes with sterile PBS solution. After adding SARS-CoV-2 S1 protein at a concentration of 5 μg/mL overnight, the membrane was washed with PBS solution five times. Next, the membrane was blocked with RPMI 1640 medium containing 10% FBS for a minimum of 30 min. Next, 5 × 10⁵ mouse splenic lymphocytes were added to each well containing RPMI 1640 medium containing 1% penicillin and incubated at 37 °C in 5% CO₂ for 24 h. Subsequently, the membrane was incubated at 37 °C. After being incubated for 24 h, primary and secondary antibodies were added and incubated separately, following the provided instructions. The ELISpot reader was used to read the spots after the color was developed with the BCIP/NTB color solution for 20 min.

Ex vivo or cultured ELISpot assays were carried out on PBMCs from HIV-positive donors. For ex vivo ELISpots, cryopreserved PBMCs were thawed and rested in R10 overnight then transferred to ELISpot plates (Merck Millipore) at 2 × 10⁵ cells per well. Peptides were added to bring the total volume/well to 200 μL; wells containing phytohemagglutinin (PHA) at 20 μg/mL concentration (Merck) or medium only were included as positive and negative controls. Plates were then incubated for 20 h at 37 °C, 5% CO₂. Coating, development (MabTech), and reading of ELISpot plates (AID Reader) have been described previously (38 (link)). For cultured ELISpots, 1.5 × 10⁶ cells were stimulated with peptide at a final concentration of 10 μg/mL in 24-well plates (Merck Millipore) with addition of 25 ng/μL interleukin (IL)-7 (R&D Systems) on day 0 in RPMI-1640 medium containing 10% human serum type AB, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM sodium pyruvate (Rab-10); 1.8 × 10³ units/mL IL-2 (R&D Systems) were added on days 3 and 7, and ELISpot assays were performed on day 10. A positive response was confirmed when the number of spots exceeded 3 times background (media only wells) and was greater than 25 spot-forming units (SFU) per million PBMCs. Zero values were not accepted in any replicate of antigen-stimulated wells.

ELISPOT assays were performed using Rat IFNγ ELISPOT Set (Abcam, Cambridge, UK) following the procedure previously described [19 (link)]. Briefly, ELISPOT plates (Merck Millipore, Darmstadt, Germany) were activated by adding 15 µL of 35% ethanol to each well. The coating with rat IFN-γ ELISPOT Capture Antibody was incubated overnight at + 4 °C. Plates were blocked with RPMI 1640 with 10% FBS for 2 h at RT. Freshly isolated rat splenocytes were plated at the concentration of 3 × 10⁵ cells/well and stimulated with 2 μg/mL S peptide (SARS-CoV-2 S1 RBD recombinant protein, RP-87706, Thermo Fisher Scientific, Waltham, MA, USA). Each condition was repeated for three technical replicates. Plates were incubated for 44 h at 37 °C with 5% CO₂. Then cells were removed, and the assay was developed following the manufacturer’s instructions. The substrate was incubated for 20 min until spot development; then, the reaction was stopped by washing with sterile water, and plates were let air-dry at RT in the dark. Plate acquisition was performed with the ImmunoSpot plate reader (S6 Macro M2, ImmunoSpot, Cleveland, OH, USA), and spot count was performed automatically by ImmunoSpot Software version 7.0.21.0 (ImmunoSpot, Cleveland, OH, USA). Data presented refer to n = 5 for rats treated with oEVs and n = 6 for rats treated with mRNA-loaded oEVs.

ELISpots were performed as previously described [15 (link)]. Briefly, ELISpot plates (Merck Millipore) were coated overnight with anti-human IFN-γ capture antibody (MabTech). Wells were washed with sterile PBS to remove excess antibody and RPMI containing 5% AB serum was added for 1 h at 37 °C. PBMC lines cultured in triplicate were pooled, washed and plated in the absence or presence of the corresponding 5T4 peptide pool or single peptide. Following 18 h of incubation at 37 °C, 5% CO₂, cells were removed, plates were washed with PBS and secondary biotinylated anti-human IFN-γ antibody was added. Wells were washed with PBS followed by the addition of streptavidin–alkaline phosphatase (Mabtech). After incubation at room temperature, wells were washed and spots were developed by the addition of a colorimetric substrate kit (Bio-Rad). The reaction was stopped after 15 min, and wells were washed in water. Spots were counted using an automated reader (Autoimmun Diagnostika GMBH, A.I.D.). Counting was checked manually using the ELISpot 5.0 software. The number of spots was normalised to 10⁵ PBMCs. Positive cultured responses were identified as having at least 25 spot-forming cells (SFC) per 10⁵ PBMC, after subtraction of the background, and an increase of at least double the number of spots above background.

To quantify antibody-secreting cells (ASCs), polyvinylidene fluoride ELISPOT plates (EMD Millipore, Burlington, MA) were coated with 200 ng/well rH1. Splenocytes and lung lymphocytes (1 × 10⁶ cells/well) were incubated at 37°C for 16 h. Influenza-specific antibodies were detected using isotype-specific, biotinylated murine Ig antibodies (IgA, IgM, and IgG; Southern Biotech; Birmingham, AL). To detect cytokine secreting cells, 5 × 10⁵ cells/well were overlaid in ELISPOT plates (EMD Millipore; Burlington, MA) coated with 100 ng/well of capture antibody (BD Biosciences; San Jose, CA). Cells were stimulated with 200 ng/well rH1 for 48 h at 37°C. The cells were washed and incubated with 100 ng/well biotinylated detection antibodies (IL-4, IL-10, IFN-γ, BD Biosciences) and developed with streptavidin-HRP and diaminobenzidine. Plates were analyzed via ImmunoSpot Reader 5.0 (Cellular Technology Limited; Shaker Heights, OH) and normalized to 1 × 10⁶ cells/well.