Pre-coated Ferret IFNγ-ELISpot (ALP) plates (Mabtech) were used according to the manufacturers protocol. Lymphocytes were stimulated with live virus (MOI 1 or 0.1) or H2N2 peptide pools in ELISpot plates at 37 °C. Per well, 250 K cells (PBMC, splenocytes), 125 K cells (nasal turbinates), and 62.5 K cells (lung lymphocytes) or undiluted cell suspension (BAL) was added. After 20 h the plates were developed according to the manufacturers protocol, with the modification that the first antibody staining was performed overnight at 4 °C. Plates were left to dry for 2–3 days after which they were packaged under BSL-3 conditions and heated to 65 °C for 3 h to inactivate any remaining infectious influenza particles. Analysis of ELISpot plates was performed using the ImmunoSpot® S6 CORE (CTL, Cleveland, OH).
Ferret ifnγ elispot alp plates
Manufactured by Mabtech
Sourced in Sweden
The Ferret IFNγ-ELISpot (ALP) plates are a laboratory tool used to detect and quantify the production of interferon-gamma (IFNγ) by ferret immune cells. The plates are coated with antibodies specific to ferret IFNγ, allowing for the capture and visualization of individual IFNγ-secreting cells through an enzymatic detection system.
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3 protocols using ferret ifnγ elispot alp plates
1
IFNγ-ELISpot Assay for Influenza Virus
2
Influenza Virus-Specific IFN-γ ELISpot Assay
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Precoated Ferret IFN-γ ELISpot (ALP) plates (Mabtech, Nacka Strand, Sweden) were used according to the manufacturer’s protocol. Lymphocytes were stimulated with live virus [multiplicity of infection (MOI) of 100 for H3N2, MOI of 1 for H5N1, and MOI of 0.1 for H1N1 and H7N9] or peptide pools (1 μg/ml) in ELISpot plates at 37°C. Per well, 250,000 cells (PBMCs), 400,000 cells (BM), 62.5,000 cells (lung lymphocytes), or undiluted cell suspension (BAL and NTs) was added. On day 56 to 2 weeks after booster vaccination, 125,000 PBMCs were used for viral stimulations because of high cellular responses. After 20 hours, the plates were developed according to the manufacturer’s protocol, with the modification that the first antibody staining was performed overnight at 4°C. Plates were left to dry for 2 to 3 days, after which they were packaged under BSL-3 conditions and heated to 65°C for 3 hours to inactivate any remaining infectious influenza particles. Analysis of ELISpot plates was performed using the ImmunoSpot S6 Core (CTL, Cleveland, OH).
3
SARS-CoV-2 IFNγ-ELISpot Assay Protocol
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Pre-coated Ferret IFNγ-ELISpot (ALP) plates (Mabtech) were used according to the manufacturers protocol. Per well, 250K PBMCs or 31.25K lung lymphocytes were stimulated with live virus (MOI 1) or SARS-CoV-2 peptide pools (1µg/peptide/ml) in ELISpot plates for 20 hours at 37°C. Plates were then washed and developed according to the manufacturers protocol, with the modification that incubation with the first antibody occurred o/n at 4°C instead of 2 hours at RT. After the final washing step, plates were left to dry for >2 days. Plates were then packaged under BSL-3 conditions and heated to 65°C for 3 hours to inactivate any remaining SARS-CoV-2 particles. Plates were analyzed on the ImmunoSpot® S6 CORE (CTL, Cleveland, OH). Spot counts were corrected for background signals by subtracting the number of spots in the medium condition from all other conditions. Data were visualized on a log-scale, so the minimum spot count was set to ‘1’ for visualization purposes.
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