To assess hIL-10R-1 binding, a competitive displacement ELISA was used. Maxisorp 96-well immunoplates (Nunc, Roskilde, Denmark) were incubated overnight with 400 ng·mL−1 of hIL-10 in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) at 4 °C. Plates were washed then blocked with 10% bovine serum albumin in PBS for 1 h. Samples of IL-10 serially diluted in PBS containing 0.4% BSA and 0.02% Tween20 were incubated with 300 ng·mL−1 hIL-10R1–Fc chimeric protein (R&D Systems, Minneapolis, MN, USA) in non-absorbent plates at 25 °C for 1 h. The mixture was transferred to plates coated with hIL-10 and incubated at 25 °C for 1 h to capture the unbound hIL-10R1–Fc protein. The captured hIL-10R1–Fc protein was detected with a goat anti-human IgG-HRP conjugate (Thermo Fisher Scientific, Waltham, MA, USA) and 3,3′,5,5′-tetramethylbenzidine substrate (BD Biosciences). The reaction was stopped with H2SO4 and quantified by measuring the absorbance at 450 nm. The percentage change in hIL-10R1-Fc protein bound to the immobilised hIL-10 was calculated relative to that detected in the absence of soluble IL-10.
3 3 5 5 tetramethylbenzidine substrate
Manufactured by BD
Sourced in United States
3,3′,5,5′-tetramethylbenzidine substrate is a colorimetric reagent used in various analytical and diagnostic applications. It serves as a substrate for enzyme-linked immunosorbent assays (ELISAs) and other enzymatic reactions, where it undergoes a color change upon catalytic conversion.
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Lab products found in correlation
Elisa plate, by Thermo Fisher Scientific (2 mentions)
Maxisorp 96 well immunoplates, by Thermo Fisher Scientific (1 mentions)
Glomax plate reader, by Promega (1 mentions)
Streptavidin conjugated horseradish peroxidase, by R&D Systems (1 mentions)
96 well plate, by Corning (1 mentions)
Enzchek protease assay kit, by Thermo Fisher Scientific (1 mentions)
Polystyrene plate, by Corning (1 mentions)
Victor3, by PerkinElmer (1 mentions)
Multiska fc microplate photometer, by Thermo Fisher Scientific (1 mentions)
Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Enspire plate reader, by PerkinElmer (1 mentions)
96 well immunoplate, by Thermo Fisher Scientific (1 mentions)
Infinite m200 microplate reader, by Tecan (1 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Synergy 2 elisa reader, by Agilent Technologies (1 mentions)
Horseradish peroxidase hrp conjugated streptavidin, by BD (1 mentions)
Recombinant human ifn γ, by BD (1 mentions)
Elx808, by Agilent Technologies (1 mentions)
Multiskan fc microplate photometer, by Thermo Fisher Scientific (1 mentions)
11 protocols using 3 3 5 5 tetramethylbenzidine substrate
1
Competitive Displacement ELISA for hIL-10R-1 Binding
2
Quantitative Analysis of Kallikrein Proteases
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For these experiments, we used primary keratinocytes cultures grown in low and high calcium conditions (see above). In KLK Elisa assays, 96-well plates (Corning, Corning, NY) were coated with rabbit anti-KLK5 or KLK7 antibody (Santa Cruz Biotechnology) at 1 μg/ml in phosphate-buffered saline (PBS). The wells were washed, blocked with PBS containing 1% BSA, and then incubated for 3 h with media isolated from keratinocytes grown for 12 hours in fresh media. Biotinylated goat anti-KLK5 or anti-KLK7 antibodies (R&D Systems) were used as detection antibodies. Colorimetric quantification at 450 nm was carried out by incubating plates with streptavidin-conjugated horseradish peroxidase (R&D Systems, Minneapolis, MN) and 3,3′5,5′-tetramethylbenzidine substrate (BD Biosciences, San Jose, CA). The reactions were stopped by 0.2 M sulfuric acid (Sigma-Aldrich, St. Louis, MO). The enzymatic activity of KLK proteins was measured by an EnzChek Protease Assay Kit (green fluorescence, Invitrogen) following the manufacturer’s recommendations. Absorbance or fluorescence was measured on a Glomax plate reader (Promega, Madison, WI)
3
Quantitative Detection of Protein Binding
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The immune enzymatic assay was used to detect specific binding of the recombinant protein rTC to anti-CD antibodies. The wells of the polystyrene plates (Costar, NY, USA) were sensitized with 8.75 ng of purified rTC dissolved in 100 µL of 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6). After incubation at 4 °C for 16 h, the coated wells were washed with PBST and blocked for 1 h at 37 °C with PBS buffer (pH 7.2) containing 5% (w/v) dried skim milk powder and washed again with PBS-T. Subsequently, 100 µL of a dilution (100 µL PBST, 5% [w/v] dried skim milk powder, and 1 µL serum) was placed into the wells, resulting in a final dilution of approximately 1:100. After incubation for 30 min at 37 °C, the wells were washed with PBST and 100 µL of goat anti-human immunoglobulin G conjugate peroxidase-labeled (Sigma Aldrich) and diluted at 1:40,000 in PBS buffer (pH 7.2), which was added following incubation for 30 min at 37 °C. The wells were again washed, and 100 µL 3,3,5,5-tetramethyl-benzidine substrate (BD Biosciences, Franklin Lakes, Nova Jersey, USA) was added and incubated for 15 min at room temperature in the dark. Reactions were stopped by the addition of 100 µL of sulfuric acid (0.2 M). The optical densities were read at 450 nm.
4
Evaluating Antigen-Specific Antibodies Post-Vaccination
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Two and four weeks after the first immunization, serum and bronchoalveolar lavage fluid (BALF) samples were analyzed for antigen (Ag)-specific IgG and IgA by enzyme linked-immunosorbent assay. ELISA plates (ThermoFisher Scientific) were coated with vaccine antigen (200 ng/well) and then incubated with the samples. After sequential incubation with peroxidase goat anti-mouse total IgG and IgA (Cell Signaling Technology, MA), 3,3′,5,5′-tetramethylbenzidine substrate (BD Bioscience, CA) was added to each well. The optical density was measured at a 450 nm wavelength by using VICTOR3™ (PerkinElmer, MA). To determine the HA stalk-specific Abs, HA419-473 from PR8 and HA379-473 from pH1N1 polypeptides were expressed in E. coli and used as coating antigens in a subsequent ELISA.
5
SARS-CoV-2 Spike Protein ELISA
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The wells of a 96-well plate pre-coated with 100 ng of S protein of SARS-CoV-2, SARS-CoV (Sino Biological Inc., Catalog Number: 40634-V08B), or MERS-CoV (Sino Biological Inc., Catalog Number: 40069-V08B) were blocked with 0.25% gelatin in PBS buffer containing 0.05% Tween-20 (PBST), and then incubated with 100 μL of indicated mAbs at room temperature for 1 h. After washing of the 96-well plate three times with PBST, the HRP-conjugated goat anti-mouse IgG (H + L) (KPL) was added to each well and incubated at room temperature for 1 h. After washing of the 96-well plate three times with PBST, 75 μL of 3,3′,5,5′-tetramethylbenzidine substrate (BD Bioscience) was added to each well for signal detection, followed by addition of 75 μL of 2 N H2SO4 to terminate the reactions. Results are evaluated quantitatively by measuring the absorbance at 450 nm by the Multiska FC Microplate Photometer (Thermo Fisher Scientific).
6
ELISA Quantification of Antibody Titers
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Maxisorp 96-well plates (Thermo Fisher Scientific) were coated with Myozime protein in carbonate buffer at 4°C overnight. A standard curve of IgG isotype (Sigma-Aldrich) was coated to the wells in seven 2-fold dilution starting from 1 μg/ml. After blocking, plasma samples diluted at 1:100 were added to plates and incubated for 1 hr at 37°C. Isotype-specific secondary antibodies coupled to HRP were used for detection (Southern Biotech). Then, 3,3′,5,5′-tetramethylbenzidine substrate (BD Biosciences) was added to the wells and color development was measured at 450 and 570 nm (for background subtraction) on an Enspire plate reader (Perkin Elmer) after blocking the reaction with H2SO4.
7
Influenza Antibody Titer Measurement by ELISA
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Influenza virus-specific antibody titers were measured on individual mouse sera by endpoint dilution ELISA as previously described by Han et al., with slight modifications [13 (link)]. Briefly, a 96-well immunoplate (Nunc, Roskilde, Denmark) was coated with influenza A/California/07/2009 (H1N1) virus at a concentration of 16 HA units/well and incubated overnight at 4°C. Following incubation, the wells were washed with PBS Tween (PBST), blocked with 1% bovine serum albumin (BSA) in PBS, and incubated with mouse serum that was 2-fold serially diluted with 1% BSA in PBS. Bound antibody was detected with horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, or IgG2a antibody for 1 h at 37°C followed by incubation with 3,3′,5,5′-tetramethylbenzidine substrate (BD Biosciences). The reaction was stopped and absorbance at 450 nm was measured using an Infinite M200 microplate reader (Tecan, Männedorf, Switzerland). The endpoint titer was defined as the highest serum dilution with an absorbance value two times greater than the nonimmune serum absorbance value, with the cut-off value set at 0.1. The geometric mean titers (GMT) were calculated from individual log10-transformed titers and were expressed as GMT ± standard deviation (SD) for each experimental group of six mice.
8
Quantifying Mouse Cytokine Levels
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Concentrations of IL-17, interferon (IFN)γ, IL-6, and IL-10 in the mouse spleen cell culture supernatants were measured using murine cytokine ELISA kits (R & D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Briefly, 96-well Maxisorp ELISA plates (Thermo Scientific, Rochester, NY, USA) were coated with the purified anti-cytokine capture Ab at the recommended concentration overnight. After repeated washing, free binding sites were blocked with a blocking buffer containing 1% bovine serum albumin (BSA; Sigma-Aldrich). Undiluted cell culture supernatants (50–100 μl/well) and serially diluted cytokine standards were then incubated in duplicate wells with the immobilized capture Ab for 2 hours. After repeated washing, biotinylated anti-cytokine detection antibody was applied to the wells followed by horseradish peroxidase (HRP)-conjugated streptavidin. The color reaction was developed with 3,3',5,5'-tetramethylbenzidine substrate (BD Biosciences, San Diego, CA, USA). Optical density (OD) at 450 nm, and corresponding cytokine concentrations were determined using a Synergy 2 ELISA reader (BioTek Instruments, Winooski, VT, USA).
9
Quantification of Plasma IgG Binding to IFN-γ
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The method has been published11 (link),17 (link)–19 (link). In brief, recombinant human IFN-γ (BD Biosciences) was coated onto ELISA plates (Maxisorp, Nunc) at 4 °C overnight while the uncoated control wells had only PBS. On the day of the experiment, a pre-coated plate was washed, and heparinized plasma added at 1:100 dilution in duplicate, and incubated for 2 h at room temperature. After washing, biotinylated mouse anti-human IgG monoclonal antibody (clone G18–145: BD Biosciences) and horse radish peroxidase (HRP) conjugated streptavidin (BD Biosciences) was added to each well. After 1 h incubation, the color was developed by adding 3,3′,5,5′-Tetramethylbenzidine substrate (BD Biosciences). The results were calculated as the absorbance index by (O.D.test – O.D.uncoated)/O.D.uncoated.
10
ELISA Quantification of IgG Antibodies
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Serum levels of IgG antibodies to LEAPS and control peptides of vaccinated mice were determined by ELISA as described [35 (link)]. In brief, biotinylated peptides (DerG-PG70, DerG-PG275Cit, PG70, PG275Cit, DerG, J, or OVA) were immobilized on Neutravidin-coated and pre-blocked plates (Pierce, Rockford, IL, USA) at 2.5 µg/well in 100 µL Tris-buffered saline. After washing, mouse serum samples at 1:1000 dilution were incubated with the immobilized peptides for 1 h at room temperature. Peptide-bound IgG antibodies were detected by incubation with horseradish peroxidase-labeled anti-mouse IgG (BD Biosciences, San Diego, CA, USA) followed by addition of 3,3′,5,5′-tetramethylbenzidine substrate (BD Biosciences) for color development. Optical density (OD) values at 450 nm were read by an ELISA reader (BioTek ELX808, Winooski, VT, USA) [21 (link)].
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