ELISA plates were coated overnight at 4 °C with 5 µg/ml of NP16bovine serum albumin (BSA) in bicarbonate coating buffer (0.1 M sodium bicarbonate and 0.02% sodium azide at pH 9.6). Plates were washed with wash buffer (PBS containing 0.05% Tween 20) and after blocking 1 hr with blocking buffer (PBS supplemented with 2% BSA and 0.05% Tween 20) at 37 °C, serially diluted serum samples were added and incubated for 1 h at room temperature. Technical duplicates were performed for every serum sample. Plates were washed with PBS with 0.05% Tween 20 and incubated with 1 µg/ml biotinylated antiIgG1b (B682, BD Biosciences) for 1 hr followed by streptavidin conjugated horseradish peroxidase for 45 min. Peroxidase activity was detected by tetramethylbenzidine substrate (Dako) and the reaction was quenched with 2 N H2SO4 and optical densities were quantified at 450 nm. The endpoint titer of each sample was determined using Prism software (GraphPad Software) from a one phase exponential decay curve defined as the dilution that generates an OD450 value of the background plus 3 standard deviations.
Tetramethylbenzidine substrate
Manufactured by Agilent Technologies
Sourced in United States
Tetramethylbenzidine substrate is a colorimetric reagent used in various analytical techniques, such as enzyme-linked immunosorbent assays (ELISA). It serves as a substrate for peroxidase enzymes, producing a blue-colored product upon oxidation. The intensity of the color change can be measured spectrophotometrically, allowing for quantitative analysis.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated antiigg1b b682, by BD (2 mentions)
Biotin conjugated goat anti mouse igg, by Merck Group (2 mentions)
Prism v 6.0g, by GraphPad (2 mentions)
Nunc maxisorp microtitre plates, by Thermo Fisher Scientific (2 mentions)
Streptavidin horseradish peroxidase, by Zymo Research (2 mentions)
Elisa max deluxe set human ifn γ kit, by BioLegend (2 mentions)
Horseradish peroxidase hrp conjugated streptavidin, by Vector Laboratories (1 mentions)
Zikv e protein, by Sino Biological (1 mentions)
Biotin labeled goat anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Np4 bsa, by Biosearch Technologies (1 mentions)
Prism software, by GraphPad (1 mentions)
9 protocols using tetramethylbenzidine substrate
1
Optimized NP-Specific ELISA Assay
2
ZIKV Protein Binding ELISA
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Polystyrene 96-well plates were coated overnight at 4 °C with 1 µg/mL of ZIKV E protein (Sinobiological) in a sodium carbonate buffer (pH 9.3). Plates were washed three times in PBS with 0.02% Tween 20 and blocked with non-fat dried milk for one hour at 37 °C with PBS, 2% BSA, and 0.02% Tween 20. Serum from hAd5-ZKV-vaccinated mice was serially diluted in PBS and then incubated at 37 °C. Plates were washed four times with PBS containing 0.02% Tween 20 and incubated with an biotin-labeled goat anti-mouse secondary antibody (Jackson Labs) for one hour. After washing and incubation with horseradish peroxidase (HRP)-conjugated streptavidin (Vector Laboratories, Burlingame, USA), plates were developed with tetramethylbenzidine substrate (Dako). The reaction was stopped with the addition of 2 N H2SO4, and emission (450 nm) was read with a microplate reader (Molecular Devices, San Jose, USA).
3
ELISA for Measuring Antibody Binding to WNV
4
ELISA for Measuring Antibody Binding to WNV
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MAb binding to WNV was measured by ELISA, as described previously27 (link). Briefly, recombinant WNV E ectodomain or DIII fragment was diluted to 5 μg/mL in 0.1 M sodium carbonate buffer (pH 9.3) and adsorbed on 96-well Nunc MaxiSorp microtitre plates (ThermoFisher Scientific) overnight at 4°C. After blocking with phosphate-buffered saline (PBS) containing 2% bovine serum albumin and 0.05% Tween 20 (PBS-BT) for 1 h at 37°C, three-fold serial dilutions of antibody in PBS-BT were incubated for 1 h at room temperature. Plates were washed with PBS plus 0.05% Tween 20 and incubated with biotin-conjugated goat anti-mouse IgG (1 μg/mL; Sigma-Aldrich SAB4600004) for 1 h at room temperature. After washing, all plates were incubated with streptavidin-horseradish peroxidase (2 μg/mL; Zymed) for 1 h at room temperature and developed with tetramethylbenzidine substrate (Dako). After the addition of 1 N H2SO4, the optical density at 450 nm was measured. Best-fit lines were fit using GraphPad Prism v 6.0g (GraphPad Software, Inc).
5
DENV-1 Neutralization and Binding Assays
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Plaque reduction neutralization tests (PRNT) and pre- and post-attachment neutralization assays were performed with DENV-1 strain 16007 on Vero cells as previously described [13] (link), [64] (link). Binding of intact MAbs or Fabs (E103, E106, and a negative control WNV E16) to DENV-1 virions (strain 16007) was detected by capture ELISA [13] (link), [64] (link). Briefly, humanized DENV-1 E50 MAb (subcomplex DIII A-strand specific antibody) was coated at 2 µg/ml on MaxiSorp (Nunc) polystyrene 96-well microtiter plates in a sodium carbonate (pH 9.3) buffer. Plates were washed three times in wash buffer (PBS with 0.02% Tween 20) and blocked for one hour at 37°C with blocking buffer (DMEM with 10% FBS). DENV-1 virions (2.5×105 PFU) diluted in DMEM with 10% heat-inactivated FBS were captured on plates for two hours at 37°C. Wells were washed thrice with blocking buffer and DENV-1 MAb or Fab was then added at 100 µg/ml and 4-fold serial dilutions to duplicate wells and incubated for two hours at 37°C. Plates were washed five times and then sequentially incubated with goat anti-mouse (whole molecule) IgG-HRP (Sigma, St Louis, MO) and tetramethylbenzidine substrate (Dako). The reaction was stopped with the addition of 2 N H2SO4 to the medium, and emission (450 nm) was read using an iMark microplate reader (Bio-Rad).
6
Quantifying C3a Levels in WNV Infection
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Anti-TNF-α or isotype control MAb treated CD11c Cre+Ifnarf/f mice were infected with WNV. At 48 hours after infection, blood was collected by intracardiac heart puncture into EDTA-coated tubes. Plasma was isolated and immediately added to microtiter plates that had been coated overnight with C3a capture antibody (4 µg/ml, BD Biosciences) and blocked with PBS and 1% BSA (Sigma) for one hour. Plates were incubated for two hours at room temperature, and after washing, C3a detection antibody (0.5 µg/ml, BD Biosciences) was added for two hours at room temperature. After washing, streptavidin-HRP (Invitrogen) was added for 30 minutes at room temperature, and the plates were developed with tetramethylbenzidine substrate (Dako) and H2SO4. The adjusted OD450 was determined by subtracting the OD450 value for each sample on blocked control wells analyzed in parallel. Titers represent the serum dilution yielding an adjusted OD450 value equivalent to three standard deviations above the background of the assay.
7
Quantifying Antigen-Specific IgG1 Titers
8
Measuring Human IFN-γ by ELISA
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Measurement of human IFN-γ in the culture supernatant was performed with ELISA MAX™ Deluxe Set human IFN-γ kit (BioLegend) according to manufacturer’s instruction. Cell-free supernatant was harvested from cell culture 24 h after coculture with effectors (E:T ratio 1:2). The plate was then washed, incubated with tetramethylbenzidine substrate (Agilent Technologies, CA), and read at 450 nm using a Synergy HT microplate reader (Biotek, VT).
9
Measuring Human IFN-γ by ELISA
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Measurement of human IFN-γ in the culture supernatant was performed with ELISA MAX™ Deluxe Set human IFN-γ kit (BioLegend) according to manufacturer’s instruction. Cell-free supernatant was harvested from cell culture 24 h after coculture with effectors (E:T ratio 1:2). The plate was then washed, incubated with tetramethylbenzidine substrate (Agilent Technologies, CA), and read at 450 nm using a Synergy HT microplate reader (Biotek, VT).
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