Tmb substrate reagent

Manufactured by BD

Sourced in United States

The TMB substrate reagent is a colorimetric reagent used in enzyme-linked immunosorbent assays (ELISAs) to detect and quantify the presence of specific proteins or other analytes. It serves as a substrate for the enzyme horseradish peroxidase (HRP), which catalyzes a reaction that produces a blue-colored product. The intensity of the color is proportional to the amount of the target analyte present in the sample.

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Lab products found in correlation

Goat anti mouse igm hrp, by Southern Biotech (2 mentions) 450 microplate reader, by Bio-Rad (2 mentions) Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions) Victor x3 light plate reader, by PerkinElmer (2 mentions) Elisa snoopers, by Avanti Polar Lipids (2 mentions) Model elx800, by Agilent Technologies (2 mentions) Elisa plate, by Corning (1 mentions) Bradford assay, by Bio-Rad (1 mentions) Anti mouse iga hrp antibody, by Southern Biotech (1 mentions) Anti mouse il 2, by BD (1 mentions) Anti il 17a, by BD (1 mentions) Anti il 10, by BD (1 mentions) Anti mouse ifn γ, by BD (1 mentions) Anti il 4, by BD (1 mentions) Pierce high sensitivity streptavidin hrp, by Thermo Fisher Scientific (1 mentions) Elisa plates, by Iwaki (1 mentions) Vmax kinetic microplate reader, by Molecular Devices (1 mentions) Igg1 hrp, by Southern Biotech (1 mentions) Igg2c hrp, by Southern Biotech (1 mentions) Igg3 hrp, by Southern Biotech (1 mentions)

Spelling variants of this product

TMB substrate (104 citations) TMB Substrate Reagent Set (77 citations) BD OptEIA TMB substrate reagent set (13 citations) TMB reagent (3 citations) BD OptEIA TMB Substrate Reagent kit (3 citations) 3,3′,5,5′-tetramethylbenzidine (TMB) substrate reagent set (2 citations) TMB Substrate Reagent A (2 citations)

28 protocols using tmb substrate reagent

Quantification of TGF-β and Bacteria-Specific IgA

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For active and total TGF-β: ELISA plates (Corning) were coated O/N at 4°C with anti-mouse/human TGF-β1 capture antibody (BioLegend), followed by blocking with 1% BSA in PBS for 2 hours at RT. Samples were treated with HCl for 10 mins and neutralized with Tris/NaOH to obtain total TGF-β. Both active and total TGF-β were detected using anti-mouse Biotin-TGF-β antibody (BioLegend), Strepdavadin-HRP (BioLegend), and TMB Substrate reagents (BD).
For bacteria specific IgA: A fecal supernatant from μMT mice was used as shown in [20 ] and the protein concentration was measured using Bradford assay (Bio-Rad). Each well of the ELISA plate was coated with 0.5 μg/ml μMT fecal supernatant. Serum was serially diluted and bacteria specific IgA was detected with anti-mouse IgA-HRP antibody (Southern Biotech).

Salem M., Wallace C., Velegraki M., Li A., Ansa-Addo E., Metelli A., Kwon H., Riesenberg B., Wu B., Zhang Y., Guglietta S., Sun S., Liu B, & Li Z. (2019). GARP Dampens Cancer Immunity by Sustaining Function and Accumulation of Regulatory T Cells in the Colon. Cancer research, 79(6), 1178-1190.

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Cytokine Profiling of Mouse Splenic CD4 T Cells

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Primary mouse splenic CD4 T cell culture supernatants were collected at designated time points and analyzed for cytokine secretion. 96-well Maxisorp plates were coated overnight at 4°C with the appropriate capture antibody (anti-mouse IFNγ, anti-mouse IL-2, anti-IL-4, anti-IL-17A, and anti-IL-10; BD Biosciences). Non-specific protein binding was prevented by blocking wells with 5% BSA in PBS for 3 h at room temperature. Culture supernatants and standards (recombinant IFNγ and IL-2, carrier-free) were diluted appropriately and added to wells. The plate was incubated overnight at 4°C, with continuous rocking. Biotinylated detection antibodies were added to wells followed by TMB substrate reagents (BD Biosciences) at a 1:1 ratio. Color development was monitored, and the reaction was terminated by the addition of stop solution (2N H₂SO₄). Absorbance was read at 450 nm using a microplate reader. Cytokine concentrations were determined relative to the standard curves generated.

Ozay E.I., Sherman H.L., Mello V., Trombley G., Lerman A., Tew G.N., Yadava N, & Minter L.M. (2018). Rotenone Treatment Reveals a Role for Electron Transport Complex I in the Subcellular Localization of Key Transcriptional Regulators During T Helper Cell Differentiation. Frontiers in Immunology, 9, 1284.

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IgE Binding Assay for rCan f 6 Allergen

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rCan f 6 and mutants were immobilised onto ELISA plates (IWAKI, Tokyo, Japan) overnight at 4 °C. After washing with PBS, the plate wells were blocked with 3% skim milk in PBS for 1 h at room temperature. Sera from 38 dog-allergic patients and 6 non-dog-allergic donors were diluted 1:250 with PBS, added to the wells (100 µL/well), and incubated for 1 h at 37 °C. Subsequently, biotin-labelled goat anti-human IgE antibody (0.5 mg/mL; Milford, MA, USA) diluted 1:5,000 with PBS was added to the wells (100 µL/well) and incubated for 1 h at 37 °C. Next, Pierce^® High Sensitivity Streptavidin-HRP (1.1 mg/mL; Thermo Fischer Scientific, Waltham, MA, USA) diluted 1:10,000 times with PBS was added (100 µL/well). For detection of allergen-IgE complexes, TMB Substrate Reagent (BD Biosciences, Bedford, MA, USA) was added (100 µL/well) and incubated for 15 min at room temperature. To stop the reaction, 1 N H₂SO₄ was used (100 µL/well). Absorbance at 450 nm was measured using the BioTek^TM Eon^TM Microplate Spectrophotometer (BioTek Instruments Inc, Winooski, VT, USA).

Yamamoto K., Ishibashi O., Sugiura K., Ubatani M., Sakaguchi M., Nakatsuji M., Shimamoto S., Noda M., Uchiyama S., Fukutomi Y., Nishimura S, & Inui T. (2019). Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4. Scientific Reports, 9, 1503.

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Mycobacterium tuberculosis Antigen Detection

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Mtb was cultured in Sauton’s synthetic medium and harvested at 20 days and 56 days. The 5 μg of the culture filtrate was coated onto plates at 4°C for 24 h and then reacted with an anti-Rv2145c mouse antibody for 1 h, followed by an enzyme-conjugated secondary antibody. TMB substrate reagent (BD) was then added for color development. The plates were read on a Vmax kinetic microplate reader (Molecular Devices Co., Sunnyvale, CA, USA) at 450 nm.

Park H.S., Back Y.W., Jang I.T., Lee K.I., Son Y.J., Choi H.G., Dang T.B, & Kim H.J. (2021). Mycobacterium tuberculosis Rv2145c Promotes Intracellular Survival by STAT3 and IL-10 Receptor Signaling. Frontiers in Immunology, 12, 666293.

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Autoantibody Profiling Using Microplate ELISA

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For specific auto-Ab, 96-well plates were precoated overnight at 4 °C with 100 µg/mL calf thymus dsDNA (D3664-5 × 2MG; Sigma-Aldrich), 5 µg/mL Sm/RNP (ATR01-10; Arotec Diagnostic Limited) or 5 µg/mL histone (ATH01-02; Arotec Diagnostic Limited). Plates were blocked for 1 h with 1% BSA in PBS before addition of diluted serum for 2 h. Specific antibodies were detected using goat anti–mouse IgM-HRP, IgG-HRP, IgG1-HRP, IgG2c-HRP, or IgG3-HRP (SouthernBiotech), and peroxidase reactions were developed using TMB Substrate Reagent (BD Biosciences), followed by quantification at 450 nm on a Microplate Reader 450 from Bio-Rad. The results are expressed in optical density (OD) units.

Beccaria C.G., Amezcua Vesely M.C., Fiocca Vernengo F., Gehrau R.C., Ramello M.C., Tosello Boari J., Gorosito Serrán M., Mucci J., Piaggio E., Campetella O., Acosta Rodríguez E.V., Montes C.L, & Gruppi A. (2018). Galectin-3 deficiency drives lupus-like disease by promoting spontaneous germinal centers formation via IFN-γ. Nature Communications, 9, 1628.

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Quantifying Mouse Immunoglobulin and Cytokine Levels

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Total IgM and IgG isotype levels were determined by ELISA in sera. Plates were coated with 10 μg/mL of the isotype-specific goat anti-mouse antibody (IgM, IgG1, IgG2c, IgG2b, and IgG3; SouthernBiotech) overnight at 4 °C and blocked with 1% BSA. Sera were incubated overnight at 4 °C. Peroxidase-conjugated anti-mouse IgM or IgG isotypes were added and incubated for 1 h at 37 °C. The reaction was developed with TMB Substrate Reagent (BD Biosciences), followed by quantification on a Microplate Reader 450 from Bio-Rad. The concentration was measured with reference to standard curves using known amounts of the respective murine immunoglobulin isotypes (SouthernBiotech).
Cytokines were determined by ELISA in mouse serum and in the supernatant of sorted splenic T cells (CD4⁺ B220⁻) from WT and Gal-3 KO mice cultured in vitro with anti-CD3 plus anti-CD28 for 72 h. ELISA was performed with paired antibodies from eBioscience for mouse IFN-γ (Cat#88-7314-77), IL-21 (Cat#88-8210-88), IL-4 (Cat#88-7044-77), IL-6 (Cat#88-7064-88) and IL-2 (Cat#88-7024-77), according to the manufacturer’s instructions.

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Plasma Anti-C5a Levels by ELISA

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Anti-C5a (4C9) levels were determined by ELISA. Briefly, mC5a (no. 2150-c5; R&D Systems, Minneapolis, MN, USA) was incubated at 4°C overnight in 96-well plates. After washing unbound protein and blocking with 2% BSA, a 1:1000 dilution of plasma was incubated overnight at 4°C. Following wash steps, the plate was sequentially incubated with biotinylated anti-mouse IgG (H and L) HRP (1:3000; BD Biosciences, San Jose, CA, USA), TMB substrate reagent (BD Biosciences) and the OD 450 nm read; 0.31 to 200 ng/mL anti-C5a (4C9) was used as a standard curve.

Toomey C.B., Landowski M., Klingeborn M., Kelly U., Deans J., Dong H., Harrabi O., Van Blarcom T., Yeung Y.A., Grishanin R., Lin J.C., Saban D.R, & Bowes Rickman C. (2018). Effect of Anti-C5a Therapy in a Murine Model of Early/Intermediate Dry Age-Related Macular Degeneration. Investigative Ophthalmology & Visual Science, 59(2), 662-673.

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Peptide-specific ELISA for Malaria Antibodies

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ELISA was performed using Maxisorp 96-well plates (Thermo scientific, Ref 442404). Plates were coated overnight at 4°C with 5 μg/mL of each peptide. Plates were blocked for 1 h at room temperature (RT) with PBS 1X-milk 3% before being incubated with primary antibodies. Human plasma from Burkina Faso, Tanzania, and Mali donors were tested at the dilution 1:50 or 1:100 in titration two fold dilution series and incubated on plate for 2 h at RT. Goat anti-human IgG conjugated to horseradish peroxidase (HRP) was used as secondary antibody at dilution 1:2,000 (Life technologies, Ref H10307) and 1:1,000 (Invitrogen, Cat No 62-8420) for 1 h at RT. Signal was revealed using TMB substrate reagent (BD OptEIA, cat 555214) for 20 min in the dark at RT, and the reaction was blocked using 1 M sulphuric acid (Merck, 1.00731.1000). Optical density (OD) was measured at 450 nm and 630 nm using a TECAN NanoQuant Infinit M200 PRO spectrophotometer.

Ayadi I., Balam S., Audran R., Bikorimana J.P., Nebie I., Diakité M., Felger I., Tanner M., Spertini F., Corradin G., Arevalo M., Herrera S, & Agnolon V. (2020). P. falciparum and P. vivax Orthologous Coiled-Coil Candidates for a Potential Cross-Protective Vaccine. Frontiers in Immunology, 11, 574330.

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Virus Binding to MARCO Receptor Assay

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Proteins were bound to EIA/RIA plates at 2.5ug/ml in sterile PBS containing calcium and magnesium, pH 7.4 (Lonza, Basel, Switzerland), washed, blocked with PBS containing 3%BSA, then incubated with VV or HIV-1 PsV diluted in DMEM + 1% FBS. Unbound virus was removed by repeated wash steps with PBS. Bound virus was detected using a rabbit polyclonal primary antibody recognizing MARCO (Abcam, Cambridge, MA) or a biotinylated human antibody recognizing HIV-1 (a kind gift from Pascal Poignard, International AIDS Vaccine Initiative, La Jolla, CA), an HRP-conjugated secondary antibody or streptavidin, respectively (Santa Cruz Biotechnology, Santa Cruz, CA), and TMB substrate reagent (BD Biosciences Pharmigen, San Diego, CA), and quantified using a microplate reader measuring absorbance at 450nm and subtracting absorbance at 570nm. Background binding (binding of the same concentrations of VV or HIV-1 PsV to plates coated with BSA) was subtracted.

MacLeod D.T., Nakatsuji T., Wang Z., di Nardo A, & Gallo R.L. (2014). Vaccinia virus binds to the scavenger receptor MARCO on the surface of keratinocytes. The Journal of investigative dermatology, 135(1), 142-150.

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Virus Binding to MARCO Receptor Assay

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