The wells of 96-well microplates were coated with wild type (WT) or chimeric VLPs (300 ng per well) and then blocked overnight at 4 °C. The wells were then incubated with two-fold serial dilutions of the antibody for 45 min at 37 °C. Antibody titers were detected using a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (diluted 1:5000 in HS-PBS, Abcam; Cambridge, UK), followed by 50 µL of 3, 3′, 5, 5′-Tetramethylbenzidine Liquid Substrate (Sigma-Aldrich, St Louis, MO) per well for 15 min at 37 °C. The reaction was stopped by the addition of 50 μl of 2 M H2SO4, and the absorbance read at 450 nm (reference, 620 nm) using an automated ELISA reader (TECAN, Männedorf, Switzerland). The median effective concentration (EC50, ng mL−1) is defined as the antibody concentration for achieving 50% binding with the antigen. Data analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA).
5 tetramethylbenzidine liquid substrate
Manufactured by Merck Group
5′-Tetramethylbenzidine Liquid Substrate is a chromogenic substrate used in various immunoassay applications to detect the presence of specific analytes. It undergoes an enzymatic reaction that produces a color change, which can be measured and analyzed to quantify the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Hs pbs, by Abcam (2 mentions)
96 well microtiter plate, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase labeled streptavidin, by Agilent Technologies (1 mentions)
L2515, by Merck Group (1 mentions)
L2630, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg antibody, by Abcam (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Epoch spectrophotometer, by Agilent Technologies (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
4 protocols using 5 tetramethylbenzidine liquid substrate
1
Antibody Binding Assay for VLPs
2
Quantifying Peptide-LPS/LTA Binding
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96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 5 µg/mL of purified LPS (from E. coli O111:B4, Sigma L2630) or LTA (from S. aureus, Sigma L2515) in PBS and then incubated for 2 h at room temperature in blocking solution (20 mM Tris-HCl pH 7.4 plus 0.05% Tween 20 and 1% BSA). Biotin-labeled peptides/proteins (2.5–20 µg/mL) were added and incubated overnight at 4°C in blocking solution. After extensive washing, bound peptides/proteins were detected by the addition of horseradish peroxidase-labeled streptavidin (1:5,000 dilution; DAKO) for 1 h at room temperature. Color was developed by adding 3,3’,5,5’-tetramethylbenzidine liquid substrate (Sigma) and optical density read at 405–620 nm.
3
Antibody Binding Affinity ELISA
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The wells of 96-well microplates were coated with WT or chimeric VLPs (300 ng per well) for 2 h at 37 °C and then blocked with 200 μL blocking solution for 2 h at 37 °C. The wells were then incubated with 100 μL of twofold serially diluted mAbs at a starting concentration of 1 μg/mL for 45 min at 37 °C. The wells were then washed and incubated with 100 μL horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (diluted 1:5000 in HS-PBS, Abcam; Cambridge, UK), followed by 50 μL of 3, 3’, 5, 5’-tetramethylbenzidine liquid substrate (Sigma-Aldrich, St Louis, MO) per well for 10 min at 37 °C and the reaction was quenched with the addition of 50 µL 2 M H2SO4. An automated ELISA reader (TECAN, Männedorf, Switzerland) was used to detect the absorbance at 450 nm (reference, 620 nm). GraphPad Prism (GraphPad Software, San Diego, CA) were used to calculate the median effective concentration (EC50, ng/mL), which is the concentration of antibody that binds to 50% of the antigen.
4
Quantification of Human sCD6 by ELISA
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ELISA detection of human sCD6 was performed on 96-well microtiter plates (Nunc, Roskilde, Denmark) coated overnight (o/n) at 4°C with PBL supernatants (SN) (100 μL). After blocking for 1 h at room temperature (RT) with PBS plus 3% BSA, biotinylated 161.8 mAb (2 μg/mL) was added and incubated for 2 h at RT. Bound mAb was detected by 1 h incubation at RT with SAv-Peroxidase (SAv-POD; Roche) and further color development by the addition of 3,3′,5,5′-tetramethylbenzidine liquid substrate (Sigma). Absorbance was measured at 450 nm with Epoch spectrophotometer (BioTek).
For immunoprecipitation of sCD6, protein G sepharose beads (GE Healthcare Life Sciences) were coupled to 161.8 mAb following manufacturer’s instructions and, then, incubated o/n at 4°C under orbital rotation with 1 mL SN from PHA- or rhIL-2-treated PBL. RshCD5 (500 ng) and rshCD6 (500 ng) were used as negative and positive controls, respectively. After washings, beads were eluted and run on 8% SDS-PAGE under reducing conditions for further Western blot analysis with rabbit anti-human CD6 antiserum (1:500) produced in our laboratory (27 (link)), followed by HRP-labeled donkey anti-rabbit Igs (GE Healthcare Life Sciences). Chemiluminescence was developed with Supersignal West Dura Extended Duration Solution (Pierce, Rockford, IL, USA) and images were obtained using ImageQuant LAS 4000 (GE Healthcare Life Sciences).
For immunoprecipitation of sCD6, protein G sepharose beads (GE Healthcare Life Sciences) were coupled to 161.8 mAb following manufacturer’s instructions and, then, incubated o/n at 4°C under orbital rotation with 1 mL SN from PHA- or rhIL-2-treated PBL. RshCD5 (500 ng) and rshCD6 (500 ng) were used as negative and positive controls, respectively. After washings, beads were eluted and run on 8% SDS-PAGE under reducing conditions for further Western blot analysis with rabbit anti-human CD6 antiserum (1:500) produced in our laboratory (27 (link)), followed by HRP-labeled donkey anti-rabbit Igs (GE Healthcare Life Sciences). Chemiluminescence was developed with Supersignal West Dura Extended Duration Solution (Pierce, Rockford, IL, USA) and images were obtained using ImageQuant LAS 4000 (GE Healthcare Life Sciences).
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