PBMCs (2 × 105 cells/well) were incubated in triplicates with 2 μg/mL of HLA-matched peptides in plates coated with the anti-human IFNγ antibody (mAb 7-B6-1; Mabtech, Stockholm, Sweden) at 37 °C for 20–24 h and developed as previously described [34 (link),35 (link)]. If the rare event when the exact HLA match was not available, we considered closely related HLA alleles within the same supertype [36 (link)]. PBMCs were first screened with peptide pools containing 10 peptides per pool and subsequently deconvoluted to identify the individual epitopes [37 (link)].
Mab 7 b6 1
Manufactured by Mabtech
Sourced in Sweden, United Kingdom
The MAb 7-B6-1 is a monoclonal antibody developed by Mabtech. It is designed to detect and quantify human IFN-gamma in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
3 amino 9 ethylcarbazole substrate, by BD (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Nbt bcip, by Thermo Fisher Scientific (1 mentions)
Streptavidin alp, by Mabtech (1 mentions)
Alkaline phosphatase alp conjugated streptavidin, by Mabtech (1 mentions)
Ifn γ elispot assay kit, by Mabtech (1 mentions)
Bcip nbt plus, by Mabtech (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
Mab 1 d1k, by Mabtech (1 mentions)
Elispot reader system, by Autoimmun Diagnostika (1 mentions)
7 protocols using mab 7 b6 1
1
ELISPOT Assay for Peptide-Specific T Cell Responses
2
Quantifying Donor-Reactive IFN-γ Secretion
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The numbers of donor‐reactive interferon (IFN) γ–secreting cells were measured by enzyme‐linked immunosorbent spot (ELISPOT). The 96‐well filtration plates (Merck Millipore, Darmstadt, Germany) were coated overnight with purified anti‐rhesus IFNγ antibodies (MT126L, Mabtech, Nacka Strand, Sweden) at 4 °C. Blocking was performed with 1% bovine serum albumin in PBS for 1 hour at room temperature, after which plates were washed with washing buffer (0.05% Tween‐20 in PBS). Splenocytes (1 × 106 cells/well) were cultured with 25Gy‐irradiated donor blood mononuclear cells for 48 hours at 37 °C in a CO2 incubator. Biotinylated anti‐rhesus IFNγ antibody (mAB7‐B6‐1, Mabtech, Nacka Strand, Sweden) was then added, followed by incubation for 1 hour at room temperature. After washing, streptavidin‐horseradish peroxide was added, followed by incubation for 1 hour at room temperature. After washing, 100 μL 3‐amino‐9‐ethylcarbazole substrate (BD Bioscience) was added to each well, and the reactions were allowed to proceed for 5 minutes. The ELISPOT plates were analyzed using an ImmunoSpotTM 3B instrument (CellularTechnologies, Cleveland, OH).
3
DENV-specific CD4+ T cell stimulation
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CD4+T cells stimulation was performed as previously described (8 (link), 15 (link)). Briefly, frozen Peripheral Blood Mononuclear Cell (PBMCs) were thawed and CD4+ T cells were isolated by magnetic bead negative selection and co-cultured with autologous APC derived from the positive selection in a 2:1 ratio. Cells were stimulated with DENV-specific pools kept at 37°C in 5% CO2, IL-2 (10 U/mL; eBioscience) was added at 4, 7, and 11 days after initial antigenic stimulation and harvested on day 14. Harvested cells were counted and plated 5 × 104 CD4+ T cells in triplicates in the presence of the HLA-matched peptide pools used for stimulation [1 μg/ml] and the individual peptide contained in the pool used for stimulation [10 μg/ml]. When the number of cells were not sufficient to test all the peptides contained in the pool used for stimulation a factorial approach has been followed, alternatively, all the peptides were directly deconvoluted after 14 days restimulation. On day 14, no additional APC were added to the culture. After 20 h of incubation at 37°C, cells were incubated with biotinylated IFNγ mAb (mAb 7-B6-1 Mabtech, Stockholm, Sweden) for 2 h and developed as previously described (7 (link), 14 (link)).
4
Quantifying T-cell responses to tumor antigens
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1 x 104 stimulated T cells were incubated with peptide-loaded dendritic cells (DCs) at 1:30 ratio on a 96 well ELISpot plates pre-coated with anti-human IFNγ antibody (mAb 1-DIK, Mabtech, UK) overnight at 37°C. DC were differentiated from autologous CD14+ monocytes, according to Miltenyi manufacturer’s protocol in ImmunoCult™ DC differentiation media (Stemcell Technologies, UK) for two weeks. DCs were loaded with 1 µg/ml CEFT, MAGED4B or FJX1 OPP. Samples were plated in duplicate or triplicate. Spots were detected with a biotin-conjugated human IFNγ antibody (mAb 7-B6-1, Mabtech UK) followed by incubation with Streptavidin ALP (Mabtech, UK) and BCIP/NBT (Thermo, UK). Plates were scanned using ImmunoSpots reader (AID, UK). The mean values were represented as spot-forming cells (SFCs) per 1 x 104 T cells. Levels were considered positive if at least two times above medium control.
5
Neoantigen-specific T Cell IFN-γ Assay
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IFN-γ production of neoantigen-specific T cells was determined using an IFN-γ ELISpot assay kit (Mabtech, Inc, OH). The effector lymphocytes (E) were added together with target cancer cells (T) in a ratio of E:T as 10:1. The 1 µg/ml biotinylated Mab 7-B6-1 (Mabtech, Inc, Cincinnati, OH) in 0.5% human AB serum was incubated at room temperature for 2 h and then 1:1000 alkaline phosphatase (ALP)-conjugated streptavidin (Mabtech, Inc) for 1 h. Then, 100 µl/well of BCIP/NBT plus (Mabtech) was added. The spots were photographed and automatically calculated by the CTL ImmunoSpot® Software (ImmunoSpot, Cleveland, OH).
6
PBMC Peptide Stimulation Assay
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Peripheral blood mononuclear cells (5 × 104) were incubated in triplicates with 0.1 ml complete RPMI 1640 in the presence of HLA-matched peptide pools and individual peptides (2 µg/ml) after 14 days of in vitro expansion. After 20 h of incubation at 37°C, cells were incubated with biotinylated IFNγ mAb (mAb 7-B6-1 Mabtech, Stockholm, Sweden) for 2 h and developed as previously described (10 (link), 13 (link)).
7
SARS-CoV-2 Peptide-Specific T Cell Response
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Stimulation was conducted with 2 × 105 PBMCs in X-VIVOTM 15 medium (Lonza) supplemented with 10% heat-inactivated AB serum and PepTivator® SARS-CoV-2 Prot_S peptide pools1 (1 µg/ml, Miltenyi Biotec). Negative control wells lacked peptides, and positive control wells included mAb CD3-2 of Kit. Cells were incubated overnight (16–20 h) at 37°C 5% CO2 in precoated anti-IFN-γ MSIP white plates (mAb 1-D1K, Mabtech). Plates were then washed five times with PBS (Sigma-Aldrich) and incubated for 2 h at room temperature with horseradish peroxidase–conjugated anti-IFN-γ detection antibody (1 μg/ml; clone mAb-7B6-1; Mabtech). After five further washes with PBS, tetramethylbenzidine substrate was added and spots were counted using an automated ELISpot Reader System (Autoimmun Diagnostika GmbH).
To quantify positive peptide-specific responses, spots of the unstimulated wells were subtracted from the peptide-stimulated wells, and the results expressed as spot-forming units (SFU)/2 × 105 peripheral blood mononuclear-stem cells (PBMCs). We determined SARS-CoV-2–specific spots by spot increment, defined as stimulated spot numbers ≥6 SFU/2 × 105 PBMCs. This cutoff was defined by calculating the mean ± 2 standard deviations of SFU/2 × 105 PBMCs in a group of healthy donors obtained prior to the start of the pandemic of SARS-CoV-2.
To quantify positive peptide-specific responses, spots of the unstimulated wells were subtracted from the peptide-stimulated wells, and the results expressed as spot-forming units (SFU)/2 × 105 peripheral blood mononuclear-stem cells (PBMCs). We determined SARS-CoV-2–specific spots by spot increment, defined as stimulated spot numbers ≥6 SFU/2 × 105 PBMCs. This cutoff was defined by calculating the mean ± 2 standard deviations of SFU/2 × 105 PBMCs in a group of healthy donors obtained prior to the start of the pandemic of SARS-CoV-2.
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