At the end of the incubation period 96 well membrane plates (Cat# MSIPS4W, Millipore, Billerica, MA) were coated with Anti-human IgG or IgM Abs diluted in PBS (Cat# SELB002 – IgG, SELB003 – IgM, R&D systems, Minneapolis, MN). After 24 hour incubation at 4°C, the plates were blocked at room temperature with PBS and 1% BSA for 2 hours. Plates were washed with PBS and 104 (newborn IgM, adult IgM and adult IgG) or 105 (newborn IgG and adult IgG) B cells were added to duplicate wells and incubated at 37°C overnight (10–14 hours). Plates were subsequently washed 3 times with PBS/0.5%Tween and then 3 times with PBS. Kit Isotype-specific detection Abs were added and further incubated at 4°C for 24 hours. Plates were washed 3 times with PBS and streptavidin HRP (Invitrogen; Carlsbad, CA, USA) was added for 30 minutes at room temperature. After washing plates with PBS, 50 μL TMB (Cat# 3651-10, Mabtech, Cincinnati, OH, USA) substrate was added. When spots were clearly developed (15 seconds to 30 seconds), plates were washed quickly with distilled water. Spots were counted using an ELISpot plate reader (CTL, Shaker Heights, OH, USA). When spot counts were below the limits of detection, a value of ½ the lower limit of detection was assigned.
3 3 5 5 tetramethylbenzidine (tmb)
Manufactured by Mabtech
Sourced in United States, Sweden
TMB is a commonly used substrate for enzyme-linked immunosorbent assays (ELISA). It is a chromogenic substrate that produces a colored product upon enzymatic reaction, allowing for colorimetric detection and quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Immunospot s4, by Cellular Technology (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Msips4w, by Merck Group (1 mentions)
Rbd mfc protein, by Sino Biological (1 mentions)
Ace2 his protein, by Sino Biological (1 mentions)
Goat anti mouse igg h l secondary antibody, by Abcam (1 mentions)
384 well plate, by Corning (1 mentions)
Immunospot 3, by Cellular Technology (1 mentions)
Pvdf membrane plates, by Merck Group (1 mentions)
Immunospot reader, by Cellular Technology (1 mentions)
Human serum, by Thermo Fisher Scientific (1 mentions)
Ifn γ elispot assay, by Mabtech (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
Goat anti human igg, by Thermo Fisher Scientific (1 mentions)
Avidin hrp, by Thermo Fisher Scientific (1 mentions)
Goat anti human igg hrp, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (1 mentions)
8 protocols using 3 3 5 5 tetramethylbenzidine (tmb)
1
Quantification of Antibody-Secreting B Cells
2
SARS-CoV-2 RBD-ACE2 Binding Inhibition Assay
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Here, 20 μl RBD-mfc protein (Sinobiological) were added to the 384-well plate (Corning) in duplets to make a final concentration of 0.2 μg/ml overnight at 4°C. Then, the plates were blocked for 1 h with blocking buffer (5% BSA plus 0.05% Tween 20). Next, 20 μl per well mouse serum with 5-fold serial dilutions were added to plates incubated at 37°C for 40 min followed by same volume with 0.2 μg/ml ACE2-his protein (Sinobiological) at 37°C for another 40 min. After washing, Goat Anti-Mouse IgG H&L secondary antibody (Abcam, ab6789, 1:10,000) was incubated with the plate at RT for 30 min. Plates were added with TMB (MabTech) and stopped with 1 mol/L HCl and followed with quantification detection. The half-maximal inhibitory concentration (IC50) was determined by using four-parameter logistic regression. The percentage of inhibition was calculated as follows: % inhibition = [(A-Blank)-(P-Blank)]/(A-Blank) × 100, where A is the maximum OD signal of RBD binding to ACE2-his when no serum was present, and P is the OD signal of RBD binding to ACE2-his in the presence of serum at a given dilution. The IC50 of a given serum sample was defined as the reciprocal of the dilution where the sample shows 50% competition. For human samples, the selection criteria were available for additional testing.
3
IgM and IgG ELISpot Assay for B Cell Analysis
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IgM and IgG enzyme-linked immunosorbent spot (ELISpot) assays (MabTech, Nacka Strand, Sweden) were performed following the instructions of the manufacturers. Briefly, 96-well polyvinylidene difluoride (PVDF) membrane plates (Millipore, Burlington, MA, USA) were coated with mouse anti-human IgM and IgG monoclonal antibodies (MabTech). Meanwhile, 3.2 × 105 resting B cells/well were seeded in 96-well round bottom microplates and incubated in the presence of the preformed complexes and 50 ng/ml IL-2, IL-6, and IL-10 (ImmunoTools, Friesoythe, Germany) at 37°C and 5% CO2 for 72 h. Then, 5 × 104 differently treated cells were placed into the wells of the PVDF plates and incubated at 37°C and 5% CO2 for 20 h. Then, biotinylated mouse anti-human IgM and IgG (MabTech, Nacka Strand, Sweden), streptavidin-conjugated HRP (MabTech), and TMB (MabTech) were added to the samples. B cells secreting IgM and IgG were assessed by an ImmunoSpot Reader (Cellular Technology, Shaker Heights, OH, USA) using ImmunoSpot 3 software (Cellular Technology). Images were prepared with PaintShop Pro.
4
Quantifying Antigen-Specific CTL Responses
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The antigen specific CTL response was determined by interferon-γ (IFN-γ) ELISpot assay according to the manufacturer’s protocol (Mabtech, Stockholm, Sweden). Briefly, 96-well plates with nitrocellulose membranes (Millipore, Bedford, MA) were pre-coated with primary anti-IFN-γ antibody (1-D1K) at 4 °C overnight. The plates were blocked with AIM-V medium containing 5% human serum (Invitrogen). Target cells (2 × 104/well) and CTL clones (2 × 103/well) were co-cultured in 200 µL of culture medium for 24 h in triplicate. These wells were treated with biotinylated secondary anti-IFN-γ antibody (7-B6-1), followed by incubation with HRP-reagent and stained with TMB (Mabtech). The spots were then quantified with automated ELISpot reader, ImmunoSPOT S4 (Cellular Technology Ltd, Cleveland, OH). Positive CTL responding specifically to the vaccinated peptide were evaluated and classified according to a previously described algorithm [29 (link)].
5
Quantifying Adenovirus-Mediated Antigen Titers
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Serial dilutions of ChAd3-5Ag were added to AD-293 (AD-293; Stratagene) cells for 1 h at 37 °C. Virus suspensions were removed and the cells were cultured for 46–48 h at 37 °C. AD-293 monolayers were washed and fixed with ice-cold methanol for 10–20 min at −20 °C. Fixed cells were stained with mouse anti-Adenovirus (Abcam, ab7428) and then incubated with goat anti-mouse IgG-HRP (Thermo Fisher). Viral foci were visualized with 3,3′,5,5′-tetramethylbenzidine liquid substrate (TMB, Mabtech). FFU were counted microscopically and ChAd3-5Ag titer was determined.
6
IFN-γ ELISpot Assay for CTL Response
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IFN-γ ELISpot assay was performed using a commercial kit (Mabtech, Stockholm, Sweden) to determine the CTL response [37, (link)38] (link). Briefly, 96-well plates with nitrocellulose membranes (Merck KGaA) were pre-coated with the primary anti-IFN-γ antibody at 4 °C overnight. After blocking with AIM-V medium containing 5% human serum, target cells (2 × 10 4 /well) and KIF20A peptidespecific CTL clones (2 × 10 3 /well) were co-cultured in 200 µL of culture medium at 37 °C for 24 h. These wells were treated with biotinylated secondary anti-IFN-γ mAb, followed by incubation with HRP-reagent and stained with TMB (Mabtech). The spots were then quantified with ImmunoSPOT S4 (Cellular Technology Ltd., Cleveland, OH, USA).
7
Transient Transfection and ELISA for Antibody Detection
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HEK293 cells were transiently transfected with heavy and light chains using Fugene HD transfection reagent (Promega). 3 days post-transfection, the supernatant was analyzed via sandwich ELISA. The following reagents were used for detecting hIgG: goat anti-human IgG for coating 1:500 in PBS (Thermo Fisher), PBS+5% BSA for blocking, and PBS+0.05% Tween-20 for washing as well as polyclonal goat anti-human IgG-HRP 1:1000 in PBS (Sigma Aldrich) and TMB (Thermo Fisher) for detection. For the detection of HBsAg-specific antibodies, pre-coated Protein G ELISA plates (Pierce, Thermo Fisher) were used. After adding samples, HBsAg-biotin was added 1:200 in PBS, and 1:500 Avidin-HRP in PBS (eBioscience) and TMB (MabTech) were used for development. Absorbance at 450 nm was measured with Tecan Reader (Tecan Group AG).
8
HBsAg-specific B cell ELISpot
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PBMC were stimulated with 1 µg/mL R848 and 10 ng/mL IL2 (MabTech) at 2x106 cells/mL for 5d at 37°C. ELISpot plates (Merck) were activated and coated ON at 4°C with 2 µg/mL HBsAg (Roche Diagnostics), washed 1x with PBS, and blocked with RPMI for 2h at RT. Stimulated PBMC were added to ELISpot plates at 1-2x105/well in RPMI and incubated for 18h at 37°C. B cells were subsequently removed, and plates were washed 5x with PBS. 1 µg/ml detection antibody MT78/145 (MabTech) was added in PBS+0.5%BSA for 2h at RT. After washing, Streptavidin-HRP (MabTech) in PBS+0.5%BSA was added for 1h at RT, followed by TMB (MabTech). Spot development was stopped by rinsing with H2O, and spot numbers were quantified with ImmunoSpot Reader (CTL Europe).
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