Cytokines were measured by ELISA with paired capture and detection Abs, anti–TNF-α Ab and anti–IL-2 Ab from eBioscience. Plates (Nunc MaxiSorp) were washed with 0.05% Tween 20 in PBS and blocked with 1% BSA in PBS. After the addition of 100 μl of 3,3′,5,5′-tetramethylbenzidine substrate solution (Sigma-Aldrich) and 100 μl of 0.1 M H2SO4, absorbance was read at 450 nm with a Laboratory Systems Multiskan Ascent plate reader.
3 3 5 5 tetramethylbenzidine substrate solution
Manufactured by Merck Group
Sourced in United States
3,3′,5,5′-tetramethylbenzidine substrate solution is a colorimetric reagent used in analytical procedures. It is a stable, ready-to-use solution that produces a color change upon reaction with certain analytes. The core function of this product is to serve as a substrate for colorimetric detection and quantification applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 2 ab, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated goat anti human igg, by Jackson ImmunoResearch (2 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Anti tnf α, by Thermo Fisher Scientific (1 mentions)
Costar 3590, by Corning (1 mentions)
6 protocols using 3 3 5 5 tetramethylbenzidine substrate solution
1
Cytokine Quantification by ELISA
2
Binding Specificity of HIV-1 Antibody
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ELISA was conducted to examine the HIV-1 Env-specific bindings in DP6-001 serum samples, EBV-transformed B cell media and the final mAb64 against gp120, gp120-Core or their D368R mutant protein as previously described 22 (link). To detect the polyreactivity of monoclonal antibodies, ELISA plate was coated with ssDNA, dsDNA and LPS and Insulin with final concentration of 10μg/ml for ssDNA, dsDNA and LPS, and 5μg/ml for Insulin in PBS, 50 μl/well, overnight at RT. After blocking for 2 hours at RT, properly diluted HmAb64 (50μl/well) were Incubated for 2 hours at RT. HRP-conjugated goat anti-human IgG (Jackson, 109-035-098) in dilution buffer (1:1000, 50 μl/well) was incubated for 1 hour at RT. At last, plate was developed for 5 minutes at 37°C in 100 µl of a 3,3’5,5’-tetramethylbenzidine substrate solution (Sigma). The reaction was stopped with 50 µl of 2N H2SO4. Plates were read at 450 nm.
3
Quantifying IL-33 and sT2 in Dendritic Cells
4
ELISA Cytokine Measurement Protocol
5
ELISA Polyreactivity Antibody Screening
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ELISA was conducted to examine the HIV-1 Env-specific bindings in DP6-001 serum samples, EBV-transformed B cell media, and the final mAb64 against gp120, gp120-Core, or their D368R mutant protein as previously described26 (link). To detect the polyreactivity of antibodies, the ELISA plate was coated with ssDNA, dsDNA and LPS and Insulin with the final concentration of 10 μg/ml for ssDNA, dsDNA, and LPS, and 5 μg/ml for Insulin in PBS, 50 μl/well, overnight at RT. After blocking for 2 h at RT, properly diluted HmAb64 (50 μl/well) were incubated for 2 h at RT. HRP-conjugated goat anti-human IgG (Jackson, 109-035-098) in dilution buffer (1:1000, 50 μl/well) was incubated for 1 h at RT. At last, the plate was developed for 5 min at 37 °C in 100 µl of a 3,3′5,5′-tetramethylbenzidine substrate solution (Sigma). The reaction was stopped with 50 µl of 2 N H2SO4. Plates were read at 450 nm.
6
IgE Immunochemical Reactivity Measurement
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The IgE immunochemical reactivity was measured according to the method described by Peñas et al. [39 (link)].
Samples were diluted to 5 μg/mL with 100 mM sodium carbonate buffer (pH 9.6). Then, 100 μL of dilution was added to each well in a 96-well polystyrene ELISA plate (Costar 3590, Corning Inc., New York, NY, USA) and incubated at 4 °C for 18–22 h. The plate was washed thrice with phosphate buffer (pH 7.0, containing 0.05% Tween 20). After drying at room temperature, 250 μL of blocking fluid (containing 2.5% bovine serum albumin) was added into each well and incubated at 37 °C for 2 h. Remove the blocking liquid and wash three times with the phosphate buffer. The plate was incubated with 100 μL of human sera (1:20 diluted with PBS containing 1% BSA, v/v) at 37 °C for 1 h. The plate was washed again and incubated with 100 μL of antihuman IgE-ε-chain-specific peroxidase (1:2000 dilution with PBS, v/v) per well. The plate was incubated at 37 °C for 1 h. The wells were washed again, and 100 μL of 3,3′,5,5′-tetramethylbenzidine substrate solution (Sigma Inc., St. Louis, MO, USA) was loaded and incubated for 20 min at 37 °C. Then, 2 M sulfuric acid was added to terminate the reaction. The absorbance was recorded at 450 nm with a BioTek μQuant microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).
Samples were diluted to 5 μg/mL with 100 mM sodium carbonate buffer (pH 9.6). Then, 100 μL of dilution was added to each well in a 96-well polystyrene ELISA plate (Costar 3590, Corning Inc., New York, NY, USA) and incubated at 4 °C for 18–22 h. The plate was washed thrice with phosphate buffer (pH 7.0, containing 0.05% Tween 20). After drying at room temperature, 250 μL of blocking fluid (containing 2.5% bovine serum albumin) was added into each well and incubated at 37 °C for 2 h. Remove the blocking liquid and wash three times with the phosphate buffer. The plate was incubated with 100 μL of human sera (1:20 diluted with PBS containing 1% BSA, v/v) at 37 °C for 1 h. The plate was washed again and incubated with 100 μL of antihuman IgE-ε-chain-specific peroxidase (1:2000 dilution with PBS, v/v) per well. The plate was incubated at 37 °C for 1 h. The wells were washed again, and 100 μL of 3,3′,5,5′-tetramethylbenzidine substrate solution (Sigma Inc., St. Louis, MO, USA) was loaded and incubated for 20 min at 37 °C. Then, 2 M sulfuric acid was added to terminate the reaction. The absorbance was recorded at 450 nm with a BioTek μQuant microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).
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