Multiscreen HTS MSIP4510 plates (Millipore, MA, United States) were preactivated with 50 µl/well of 70% ethanol, washed with sterile water, and coated overnight with either 500 ng/well of recombinant F protein (Sinobiologicals, Beijing, China) diluted in 50 mmol/l NaHCO3 coating buffer, or with 150 ng/well of anti-IgG or anti-IgA capture mAbs diluted in PBS as a positive control. After washing, five times with PBS, plates were blocked with R10 (RPMI, 10% FBS, 200 mmol/l L-Glutamine, 50µg/ml Streptomycin, 50U Penicillin, 1M HEPES) culture medium for 30 minutes at room temperature. Freshly isolated PBMC were plated in R10 at 500,000 and 250,000 cells/well in duplicate (250,000/well and 125,000/well for total IgA and IgG, respectively) and left overnight at 37°C, 5% CO2. Plates are developed by subsequent incubation with biotinylated anti-IgG and anti-IgA detection mAbs, followed by streptavidin–alkaline phosphatase conjugate and finally with BCIP/NBT-plus till distinct spots emerged. All antibodies and ELISpot reagents were from Mabtech, Sweden. Plates were analyzed by an A.EL.VIS automated plate reader and responses were expressed as the number of antigen-specific IgG or IgA ASC per million PMBCs.
Elispot reagents
Manufactured by Mabtech
Sourced in Sweden
ELISpot reagents are a set of laboratory materials used to detect and quantify specific cells that produce cytokines or other secreted proteins. The reagents enable the analysis of the frequency and function of individual antigen-specific cells in a sample.
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5 protocols using elispot reagents
1
ELISpot Assay for Antigen-Specific IgG and IgA
2
Mouse T Cell ELISPOT Quantification
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Mouse T cell ELISPOT reagents were obtained from Mabtech (Cincinnati, OH). Antigen specific IFN-γ+ and IL-2+ T cells were quantified per manufacturer’s instructions. Positive control wells were stimulated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) and 500 ng/mL ionomycin (Sigma-Aldrich, St. Louis, MO). Test splenocyte wells were stimulated with the appropriate peptides at a concentration of 10 μg/mL. Cells were incubated for 20 h at 37°C in 5% CO2. Positive spots were visualized on a CTL Imager and counting was performed with Immunospot software (Cellular Technology, Shaker Heights, OH). Splenocytes from VEEV vaccinated mice were stimulated with pooled 15-mer peptides containing an 11-base overlap spanning either the VEEV IAB E1 or E2 envelope glycoprotein (Pepscan, Lelystad, Netherlands). Splenocytes from EBOV vaccinated mice were stimulated with pooled 15-mer peptides containing a 10-base overlap spanning the envelope glycoprotein of EBOV (Mimotopes, Victoria, Australia).
3
Cytokine-Specific ELISpot Assay for Simian T-cell Responses
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Enzyme-linked immunospot (Elispot) assays were performed for Th1 (IFN-γ, IL-2) and Th2 (IL-4) cytokines using commercially available monkey’s IFN-γ, IL-2 and IL-4 ELISpot reagents (Mabtech AB, Nacka Strand, Sweden), as previously described [26] (link). Briefly, PBMCs (2×105) were seeded in 96-well multiscreen plates (Millipore Corporation, Billerica MA, USA), previously coated with mAb against simian IFN-γ, IL-2 or IL-4, in the presence of: i) medium alone (negative control), ii) PHA (2 µg/ml) (positive control for cytokine release) or iii) a pool of all Tat peptides (2 µg/ml of each peptide), in duplicate wells. Spots were counted using an automated ELISpot reader (A.EL.VIS, Hannover, Germany). Results are expressed as number of spot forming cells (SFC)/106 cells after subtraction of the background (the number of SFC/106 cells detected in the unstimulated sample). The cut-off values were calculated as the mean number of SFC/106 cells (+2 SD) determined in PBMC cultures from naïve monkeys stimulated with the Tat peptides upon subtraction of the background. Fold-increase over the background was also calculated. According to both criteria, samples yielding for IFN-γ ≥80 SFC/106 cells and a fold increase ≥2.5, for IL-2≥30 SFC/106 cells and a fold increase ≥3, for IL-4≥20 SFC/106 cells and a fold increase ≥2.5, were considered positive.
4
T Cell ELISPOT for Measuring CCHFV-Specific Immunity
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Mouse T cell ELISPOT reagents were obtained from Mabtech (Mabtech). Antigen-specific IFN-γ+ and IL-2+ T cells were quantified per manufactures instructions. Positive control wells were stimulated with 10 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich). Test splenocyte wells were stimulated with the appropriate peptides at a concentration of 2.5 µg/ml. Cells were incubated for 20 h at 37 °C in 5% CO2. Positive spots were visualized on a CTL Imager and counting was performed with Immunospot software (Cellular Technology Ltd.). Splenocytes from vaccinated mice were stimulated with pooled 15-mer peptides (9 pools of 17 peptides and 1 pool of 15 peptides) containing a 5-base overlap spanning either the CCHFV-IbAr 10200 or CCHFV-Afg09-2990 M-segment open reading frames (Mimotopes).
5
T-cell ELISpot Assay for Antigen-Specific Responses
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Mouse T-cell ELISpot reagents were obtained from Mabtech. Antigen specific IFN-γ+ and IL-2+ T cells were quantified per manufacturer's instructions. Positive control wells were stimulated with 10 ng/mL PMA (Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich). Test splenocyte wells were stimulated with the appropriate peptides at a concentration of 2.5 µg/mL as previously described (Suschak et al., 2021 (link)). Cells were incubated for 20 h at 37 °C in 5% CO2. Positive spots were visualized on a CTL Imager and counting was performed with Immunospot software (Cellular Technology Ltd.). Splenocytes from vaccinated mice were stimulated with pooled 15-mer peptides (2 pools of 17 peptides) containing a 5-base overlap in previously identified T-cell dominant regions of the Afg09-2990 M-segment open reading frames (Mimotopes).
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