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Tmb substrate solution

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TMB substrate solution is a laboratory reagent used as a chromogenic substrate in various immunoassay techniques. It is commonly used in enzyme-linked immunosorbent assays (ELISAs) to produce a colored reaction that can be measured colorimetrically, indicating the presence or concentration of a target analyte.

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Lab products found in correlation

Versamax microplate reader, by Molecular Devices (1 mentions) Mouse specific elisa kit, by R&D Systems (1 mentions) Clone 45m1, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase hrp conjugated streptavidin, by BD (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Duoset elisa kit, by R&D Systems (1 mentions) Elisa reader, by BMG Labtech (1 mentions) 96 well microtiter plate, by Corning (1 mentions) Mouse ige elisa kit, by BD (1 mentions) Maxisorp, by Thermo Fisher Scientific (1 mentions) Hrp conjugated anti mouse antibody, by Jackson ImmunoResearch (1 mentions) Victor x4, by PerkinElmer (1 mentions) Hmw ha, by Merck Group (1 mentions) Covalink plates, by Thermo Fisher Scientific (1 mentions) Anti human igg hrp, by Bio-Rad (1 mentions) Maxisorp plate, by Thermo Fisher Scientific (1 mentions) 96 well plate, by Thermo Fisher Scientific (1 mentions) Elisa plate reader, by Bio-Rad (1 mentions) Celltiter glo assay, by Promega (1 mentions) Mcpt1, by Thermo Fisher Scientific (1 mentions)

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TMB substrate (104 citations) TMB solution (5 citations)

23 protocols using tmb substrate solution

1

Evaluating Antigen-Specific Antibody Levels

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The levels of antibodies specific to protamine, Dex40-GTMAC2, and Dex40-GTMAC3 were evaluated using standard indirect ELISA. Briefly, wells of a 96-well plate (Nunc MaxiSorp) were coated O/N at RT with 50 μl of antigen solution (25 μg·ml-1 in PBS). The wells were blocked O/N with 200 μl of 1% BSA in PBS at 4°C. Serum samples were diluted in PBS, added to the wells washed with PBS, and incubated O/N at 4°C. Murine antibodies bound to the antigen were detected with horseradish peroxydase-conjugated secondary antibodies specific to mouse IgM (Sigma A8786, 1:5 000) or mouse IgG (Sigma A3673, 1:10 000). Colorimetric detection was based on TMB substrate solution (BD Biosciences) and the enzymatic reaction was stopped by adding 50 μl of 0.18 M H2SO4. ELISA signals were measured at 450 nm using VERSAmax microplate reader (Molecular Devices). All sera were tested on each antigen (protamine, Dex40-GTMAC2, and Dex40-GTMAC3) to detect potential cross-reactivity.
Kalaska B., Kaminski K., Sokolowska E., Czaplicki D., Kujdowicz M., Stalinska K., Bereta J., Szczubialka K., Pawlak D., Nowakowska M, & Mogielnicki A. (2015). Nonclinical Evaluation of Novel Cationically Modified Polysaccharide Antidotes for Unfractionated Heparin. PLoS ONE, 10(3), e0119486.
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2

Quantifying Pulmonary Inflammatory Markers

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BAL supernatants (25 μl) were used to determine neutrophil myeloperoxidase (MPO) using a mouse-specific ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer instructions. Secreted MUC5AC protein was also determined in BAL supernatants following the published method 29 (link). Briefly, an aliquot of BAL supernatant (20 μl) was loaded in each well of an ELISA plate containing a goat polyclonal anti-MUC5AC capture antibody (1:40 dilution of sc-16903; Santa Cruz Biotechnology Inc., Dallas, TX) in pH 9.5 bicarbonate-carbonate coating buffer (BD OptEIA Reagent; BD Biosciences, San Diego, CA). The plate was incubated at 48°C until the reaction was dry (>5 hr). The wells were washed and blocked overnight with an assay diluent containing 10% fetal bovine serum (BD Opt EIA) at 4°C. The samples were then incubated with a biotinylated monoclonal anti-MUC5AC detection antibody (1:100 of Clone 45M1, Thermo Scientific, Waltham, MA) for 1.5 hr at 37°C. Following incubation with a peroxidase-conjugated secondary antibody (1:2500, goat anti-mouse-IgG-HRP), color change was developed by adding the TMB substrate solution (BD Biosciences). Optical density was measured at 450 nm after the stop buffer was added.
Cho H.Y., Park S., Miller L., Lee H.C., Langenbach R, & Kleeberger S.R. (2021). Role for mucin-5AC in upper and lower airway pathogenesis in mice. Toxicologic pathology, 49(5), 1077-1099.
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3

ELISA Quantification of Allergy Markers

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IL-4, IL-13, and IFN-γ levels in splenocyte cultures were determined by ELISA Duoset kit (R&D System Inc., Minneapolis, MN, USA), and IL-5 levels were measured by mouse IL-5 ELISA kit (BD PharMingen, San Diego, CA, USA), according to the manufacturer's instructions. For the levels of OVA-specific IgE antibodies, 10 μg/ml of OVA was coated onto the plates (Costar, Corning, NY, USA), blocked by 1% bovine serum albumin (BSA) (Sigma-Aldrich), and then incubated with serum samples. Next, biotinylated rat anti-mouse IgE monoclonal antibodies (BD PharMingen), streptavidin-conjugated horseradish peroxidase (HRP) (BD PharMingen), and TMB substrate solution (BD PharMingen) were sequentially added to the plates. The reaction was stopped with 2 N H2SO4. Absorbance was measured on an ELISA reader at 450 nm (SpectraMax M2 Molecular Devices, CA, USA).
Ma J., Liu M.X., Chen L.C., Shen J.J, & Kuo M.L. (2021). Ding Chuan Tang Attenuates Airway Inflammation and Eosinophil Infiltration in Ovalbumin-Sensitized Asthmatic Mice. BioMed Research International, 2021, 6692772.
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4

ELISA for Influenza Virus Antibody Detection

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ELISA was performed to analyze whether the vaccine-induced antibodies specifically bind to the whole influenza virus or protein antigen. Ninety-six-well plates (SPL, Korea) were coated with 100 μL of 105 PFU/well whole viruses or 100 μL of 0.1 μg/well of proteins overnight at 4°C. The plates were washed three times with 120 μL/well of PBST (0.05% Tween20 in PBS, pH 7.5) and blocking with 150 μL/well of blocking buffer (1% [w/v] BSA in PBST) for 1 h at room temperature (RT). Then, the plates were incubated with 100 μL/well of two-fold serial diluted mouse sera or nasal turbinate samples for 1 h at RT. After washing three times, the plates were incubated with 100 μL/well of 1:10,000 or 1:5,000 diluted horse-radish peroxidase (HRP)-conjugated secondary goat anti-mouse IgG1, IgG2a, or IgA (Bethyl, US) for 1 h at RT. Then, the plates were washed and incubated with 100 μL/well TMB substrate solution (BD Biosciences, UK) for 30 min at RT. After color development, 50 μL/well 2N of sulfuric acid (H2SO4) solutions was added to stop the reaction, and the optical density at 450 nm (OD450nm) was measured on an ELISA reader (BMG Labtech, Germany).
Kim M., Cheong Y., Lee J., Lim J., Byun S., Jang Y.H, & Seong B.L. (2021). A Host-Restricted Self-Attenuated Influenza Virus Provides Broad Pan-Influenza A Protection in a Mouse Model. Frontiers in Immunology, 12, 779223.
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5

Serum IgE Quantification by ELISA

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Blood samples were collected by centrifugation at 2500 g for 20 min. The serum was diluted (1:250) with 5% fetal bovine serum (FBS) in PBS (assay diluent) and the serum IgE levels were measured using a mouse IgE ELISA kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, a 96-well microtiter plate (Costar, NY, USA) was coated overnight at 4 °C with anti-mouse IgE mAb. After wash with PBS containing 0.05% Tween 20, the plates were blocked with 5% FBS in PBS for 1 h at RT. The diluted 100 µL serum samples were incubated for 2 h at RT. Secondary peroxidase-labeled biotinylated anti-rat IgE mAb was incubated in blocking buffer for 1 h. The enzyme reaction was initiated by addition of TMB substrate solution (BD Biosciences) for 30 min, and the reaction was stopped by the addition of 50 µL stop solution to each well. The optical density was measured at 450 nm with a microplate reader (SOFT max PRO, version 3.1. Molecular Devices, Sunnyvale, CA, USA). The lower limit of detection for the IgE ELISA was 1.5 ng/mL.
Jung K.H., Baek H., Kang M., Kim N., Lee S.Y, & Bae H. (2017). Bee Venom Phospholipase A2 Ameliorates House Dust Mite Extract Induced Atopic Dermatitis Like Skin Lesions in Mice. Toxins, 9(2), 68.
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6

Quantification of Immunoglobulin Levels

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Cell-free coculture supernatants were collected and stored at −80°C. The concentrations of immunoglobulins were measured by ELISA. In brief, 96-well plates (MaxiSorp, Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at 4°C with 10 µg/ml mouse monoclonal anti-human IgG, IgA or IgM (AbD Serotec, Munich, Germany), and subseqently blocked with 2% BSA/PBS. Standard curves of human IgG, IgA or IgM (Sigma-Aldrich) together with culture supernatants diluted in 2% BSA/PBS were incubated for 3 hours at room temperature, washed and developed with HRP-conjugated goat anti-human IgG, IgA or IgM (ABD serotec) followed by TMB substrate solution (BD-Pharmingen). Absorbance was measured at 450 nm in a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTec Instruments, Inc., Winoosi, VT, USA).
Bautista-Caro M.B., Arroyo-Villa I., Castillo-Gallego C., de Miguel E., Peiteado D., Plasencia-Rodríguez C., Villalba A., Sánchez-Mateos P., Puig-Kröger A., Martín-Mola E, & Miranda-Carús M.E. (2014). Decreased Frequencies of Circulating Follicular Helper T Cell Counterparts and Plasmablasts in Ankylosing Spondylitis Patients Naïve for TNF Blockers. PLoS ONE, 9(9), e107086.
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7

Quantifying Antigen-Specific Antibody Levels

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Anti-OVA polyclonal rabbit antibody (#ABIN400491; Antibodiesonline) was diluted in carbonate–bicarbonate buffer at 1:2,000, coated overnight at 4°C on MediSorp 96-well plates (ThermoFischer), and washed with PBS-0.05% Tween 20. Plates were incubated during 1 hour with PBS-5% bovine serum albumin, and samples were added for 2 hours at room temperature after washing. Samples were either supernatants collected from 0.5×106 plated cells 2 days after transfection with Gag-OVA, OVA-C1C2, or mock plasmids and treated with Triton-X100 detergent; or the pellets obtained after EV purification, treated or not with Triton-X100. Bound OVA was revealed by polyclonal mouse anti-OVA (#ABIN316446, at 1:2,000), followed by HRP-conjugated anti-mouse antibodies (Jackson Immunoresearch, 115-035-166) revealed by TMB-substrate solution (BD OptEIA). Reaction was stopped with 1 N HCl, and absorbance was read at 450 nm.
Sedlik C., Vigneron J., Torrieri-Dramard L., Pitoiset F., Denizeau J., Chesneau C., de la Rochere P., Lantz O., Thery C, & Bellier B. (2014). Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms. Journal of Extracellular Vesicles, 3, 10.3402/jev.v3.24646.
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8

Quantification of hCLEC14a-CTLD-Fc-HRP Binding

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Human umbilical vein endothelial cells (1 × 104) were grown in 0.1% (w/v) gelatin‐coated wells of a 96‐well plate overnight at 37 °C. Following fixation with 4% (w/v) PFA, the cells were incubated with 3 μg·mL−1 hCLEC14a‐CTLD‐Fc‐HRP in the presence or absence of increasing concentrations of 20 μg·mL−1 deglyco C1 IgG for 2 h at 37 °C. After three washes with ice‐cold PBS, 100 μL of TMB substrate solution (BD Biosciences) was added to each well. The reaction was stopped by the addition of an equal volume of 1 N H2SO4. The optical density was measured at 450 nm using a spectrophotometer (VICTORTM™ X4; PerkinElmer).
Kim T., Park C.S., Jang J., Kim M.R., Na H., Lee K., Kim H.J., Heo K., Yoo B.C., Kim Y., Lee J., Kim S.J., Kim E.S., Kim D.Y., Cha K., Lee T.G, & Lee S. (2018). Inhibition of VEGF‐dependent angiogenesis and tumor angiogenesis by an optimized antibody targeting CLEC14a. Molecular Oncology, 12(3), 356-372.
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9

Quantitative BALF Cytokine Profiling

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The levels of IL‐4, IL‐5, IL‐13, and IFN‐γ in the BALF were assessed using a quantitative sandwich enzyme‐linked immunoassay kit (BD Biosciences for IL‐4, IL‐5, IFN‐γ, and R&D Systems, Minneapolis, MN, for IL‐13). A 96‐well‐plate was coated overnight at 4°C with anti‐mouse IL‐4, IL‐5, IFN‐γ, or IL‐13 mAbs in coating buffer. After washing, the wells were blocked with 5% FBS in PBS and 1% BSA in PBS for 1 h at 4°C and RT, respectively. Subsequently, the wells were loaded with 100 μl of BALF and incubated for 2 h at RT. After washing, the secondary peroxidase‐labeled biotinylated anti‐mouse IL‐4, IL‐5, IFN‐γ, or IL‐13 mAbs in assay diluents were added for 1 h. Finally, the plates were treated with TMB substrate solution (BD Biosciences) for 30 min, and the reaction was stopped by the addition of 50 μl of TMB stop solution per well. The optical density was measured at 450 nm using a microplate reader (SOFT max PRO, version 3.1. software, Sunnyvale, CA, USA). All of the results were normalized to the total amount of BALF protein in each sample.
Park S., Baek H., Jung K., Lee G., Lee H., Kang G., Lee G, & Bae H. (2015). Bee venom phospholipase A2 suppresses allergic airway inflammation in an ovalbumin‐induced asthma model through the induction of regulatory T cells. Immunity, Inflammation and Disease, 3(4), 386-397.
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10

Quantifying Siglec-Glycosaminoglycan Interactions

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The binding of hSiglec-9/5/7/11 and hCD44–Fc chimeras to HMW-HA (Sigma-Aldrich) was determined using a previously described method with minor modifications. Briefly, 10 µg/well HMW-HA was covalently bound to CovaLink plates (Thermo Scientific) using 1% EDC (1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride) (Thermo Scientific). Plates were incubated for 2 h at 37°C and then overnight at room temperature. Wells were blocked with 1% BSA/PBS for 2 h at room temperature. hSiglec-9–Fc was diluted in 20 mM Tris (pH = 8.0), 150 mM NaCl, 1% BSA at 0.125 µg/well and incubated for 2 h at 37°C. Anti-human IgG-HRP (Biorad) was used as secondary antibody at 1:5,000 dilution and incubated for 1 h at 37°C. TMB substrate solution (BD Biosciences) was added and the absorbance was detected at 450 nm.
Secundino I., Lizcano A., Roupé K.M., Wang X., Cole J.N., Olson J., Ali S.R., Dahesh S., Amayreh L.K., Henningham A., Varki A, & Nizet V. (2015). Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation. Journal of molecular medicine (Berlin, Germany), 94(2), 219-233.
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