Analysis of SARS-CoV-2-specific memory B cells was performed by FluoroSpot assay detecting receptor-binding domain (RBD)-specific memory B cell following polyclonal B cell stimulation. We used reversed antigen human IgG SARS-CoV-2 RBD ELISpotPLUS (Mabtech, Sweden) according to manufacturer instructions. Briefly, PBMCs were incubated (250,000 cells/well) on an anti-IgG FluoroSpot plate after stimulation with a mixture of R848 (1μ/ml) and IL2 (10 ng/ml) B-Cell stimpack (Mabtech, Sweden) (Jahnmatz et al., 2020 ). The number of SARS-CoV-2-specific IgG secreting B cells was measured as Spot-Forming Units (SFU) using Mabtech IRIS™ reader. The results were expressed as the number SFU per 250,000 seeded cells after subtracting the background of unstimulated cells. Positive cutoff value was set above the 90% confidence interval in healthy non-vaccinated subjects (n = 10, cutoff = 5.0 SFU).
Iris reader
Manufactured by Mabtech
Sourced in Sweden
The IRIS™ reader is a laboratory instrument designed for the analysis of ELISA (Enzyme-Linked Immunosorbent Assay) plates. It is capable of measuring the absorbance of samples in the microplate wells, which is a common method for quantifying the presence of specific analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
B cell stimpack, by Mabtech (3 mentions)
Rbd elispotplus, by Mabtech (2 mentions)
Akp streptavidin, by BD (1 mentions)
96 well filtration plate, by Merck Group (1 mentions)
Biotin anti human igg, by BD (1 mentions)
Interleukin 7 (il 7), by R&D Systems (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
6 protocols using iris reader
1
Quantifying SARS-CoV-2-Specific Memory B Cells
2
SARS-CoV-2 Specific Memory B-cell Analysis
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Analysis of SARS-COV-2 specific memory B-cells was performed by FluoroSpot assay detecting receptor-binding domain (RBD) memory B cell following polyclonal B cell stimulation. We used reversed antigen human IgG SARS-COV-2 RBD ELISpotPLUS (Mabtech, Sweden), according to the manufacturer instructions. Briefly, peripheal blood mononuclar cells (PBMCs) were incubated (250,000 cells/well) on an anti-IgG FluoroSpot plate after stimulation with a mixture of R848 (1mkg/ml) and IL2 (10 ng/ml) (B-Cell stimpack, Mabtech, Sweden). The number of SARS-COV-2 specific IgG secreting B-cells was measured as Spot Forming Units (SFU) using Mabtech IRIS™ reader. The results were expressed as the number SFU per 250,000 seeded cells after subtracting the background of unstimulated cells. Positive cut-off value was set above the 90% confidence interval in healthy non-vaccinated subjects, (n = 10, cutoff = 5.0 SFU), Suppl Fig. 1 A.Fig. 1 ![]()
Post-vaccination COV-2 IgG antibody titer by DMTs in relation absolute lymphocyte count presented as grading >1500 cells/mm3 (green circles), between 1000 and 1500/cells mm3 (purple), between 500 and 1000/cells mm3 (orange), <500/mm3 (red), no data (grey). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3
ELISpot Assay for PS-specific IgG-secreting Cells
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For the ELISpot assay of PS-specific IgG-secreting cells in humans and mice, a 96-well filtration plate (Millipore, Billerica, MA, USA) was coated with 1 mg/ml phosphatidylserine-BSA antigen (Cloud-Clone) (100 μl/well) at 4 degrees centigrade and incubated overnight. Then, purified B-cell subsets (2000 cells per well) were cultured in antigen coating filtration plates for 48 h. Then, biotin anti-human IgG (Clone: G18-145, BD Pharmingen) and biotin anti-mouse IgG (Clone: Poly4053, BioLegend) were added as detection antibodies. AKP streptavidin (BD Biosciences) and NBT/BCIP substrate (Millipore) were used. Finally, PS-specific IgG-secreting cells were quantified. To measure and compare the antibody-secreting capacity of various B-cell subsets, we performed separate experiments using sorting-purified B1a, B1b and B2 cells from the peritoneal cavity of 10-week-old female MRL/Lpr mice in the ELISPOT assay and measured the parameters, including the spot number as spot forming units (SFU) and the spot size as relative spot volume (RSV), using an IRIS reader (Mabtech, Sweden) [24 (link)].
4
Quantification of T-cell Responses
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Every 3 weeks, 45 mL peripheral blood was drawn into EDTA tubes and peripheral blood mononuclear cells (PBMC) were isolated using Ficoll gradient centrifugation for each subject. PBMCs were thawed in RPMI1640 with 10% FBS and incubated for 7 days at 37°C, 5% CO2. On day 1 and 4, the medium was refreshed and supplemented with 5 ng/mL IL7 or 5 ng/mL IL7 and 4 ng/mL IL2 (R&D Systems), respectively. After 7 days, PBMCs were harvested and rested for 16 hours, then seeded in 2 or 3 replicates (depending on sample availability) on IFNγ/Granzyme-B/TNFα FluoroSpot plates (Mabtech) at 100,000 to 150,000 cells/well in RPMI-10% FBS. Cells were incubated either with the RPMI-10% FBS medium, or the peptides (5 μg/mL), or peptide pools (5 μg/mL per peptide) overnight at 37°C, 5% CO2. Development was performed according to the manufacturer's instructions. Results were acquired with a Mabtech IRIS reader and analyzed using the Mabtech Apex software. In vitro stimulated (IVS) ELISpot results were considered positive when after subtracting the corresponding nonstimulated control (ΔSFU), the result was >2.5-fold higher than the DMSO negative control. A response was considered “boosted” compared with pre-vaccination when at least two-fold increase in ΔSFU was achieved.
5
Memory B Cell Response to SARS-CoV-2 RBD
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Analysis of SARS-CoV-2-specific memory B cells was performed by FluoroSpot assay detecting receptor-binding domain (RBD)-specific memory B cell following polyclonal B cell stimulation. We used reversed antigen human IgG SARS-CoV-2 RBD ELISpotPLUS (Mabtech, Sweden) according to manufacturer instructions. Briefly, PBMCs were incubated (250,000 cells/well) on an anti-IgG FluoroSpot plate after stimulation with a mixture of R848 (1 mkg/ml) and IL2 (10 ng/ml) B-Cell stimpack, Mabtech, Sweden. The number of SARS-COV-2-specific IgG secreting B cells was measured as Spot-Forming Units (SFU) using Mabtech IRIS™ reader. The results were expressed as the number SFU per 250,000 seeded cells after subtracting the background of unstimulated cells. Positive cutoff value was set above the 90% confidence interval in healthy non-vaccinated subjects (n = 10, cutoff = 5.0 SFU).
6
Quantifying Anti-RBD Memory B Cells
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Anti‐RBD‐specific MBC were detected in PBMCs by the ELISpot assay. PBMCs were cultured for 5 days in RPMI‐1640 medium with 10% fetal bovine serum and 1% penicillin–streptomycin, supplemented with 1 μg/ml R848 (resiquimod; a TLR7/8 agonist) and 10 ng/ml recombinant human IL‐2. After incubation cells were washed and a total of 250.000 PBMC per well were loaded onto ELISpot plates pre‐coated with monoclonal anti‐human IgG antibodies for 24 h (37°C, 5% CO2). The human IgG SARS‐CoV‐2 RBD ELISpotPLUS (ALP kit, Mabtech, Sweden) was used according to the manufacturer's instructions as described previously.6 The number of MBC was measured as spot forming units (SFU) using a Mabtech IRIS™ reader. The results were expressed as SFU per 250.000 PBMC. The positive cut‐off value (5.0 SFU/250.000 PBMC) was calculated as a 90% confidence interval in 16 adult healthy controls.
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