Tmb elisa peroxidase substrate

Manufactured by Rockland Immunochemicals

Sourced in United States

TMB ELISA peroxidase substrate is a chromogenic substrate used in enzyme-linked immunosorbent assays (ELISAs) to detect and quantify the presence of target analytes. It serves as a substrate for the horseradish peroxidase (HRP) enzyme, which catalyzes a reaction that produces a colored product, indicating the presence of the target analyte.

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Lab products found in correlation

Synergy ht multi mode microplate reader, by Agilent Technologies (3 mentions) Streptavidin hrp, by Southern Biotech (2 mentions) Tristar lb 941, by Berthold Technologies (2 mentions) Hrp conjugated goat anti mouse secondary antibody, by Southern Biotech (2 mentions) Goat anti mouse ig, by Southern Biotech (2 mentions) Anti igg horseradish peroxidase conjugated secondary antibody, by Southern Biotech (1 mentions) M9410, by Merck Group (1 mentions) Elx808 absorbance plate reader, by Agilent Technologies (1 mentions) Immunoblock, by Sumitomo Pharma (1 mentions) Hrp conjugated goat anti mouse igm, by Southern Biotech (1 mentions) Elisa plates, by Merck Group (1 mentions) Igg2c, by Southern Biotech (1 mentions) Igg2b, by Southern Biotech (1 mentions) Igg2a, by Southern Biotech (1 mentions) Dve00, by R&D Systems (1 mentions)

9 protocols using tmb elisa peroxidase substrate

Cytomegalovirus Seroprevalence Assessment via ELISA

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As described by Kilgour et al, CMV ELISA testing (University of Birmingham, Birmingham UK) was used to ascertain participant’s CMV status. Briefly, mock and viral lysate were used to coat 95 well plate overnight at 4 °C. Using plasma from 3 CMV positive donors, a standard was prepared and added to the plate in a 1 in 4 serial dilution, alongside 1ul of healthy donor samples. After 1 h incubation at room temperature, the plate was washed and anti-IgG horseradish peroxidase conjugated secondary antibody (Southern Biotech, Alabama, USA) added to each well and incubated for a further 1 h in the dark, RT. After repeat wash steps, 100 μL of TMB ELISA peroxidase substrate (Rockland Immunochemicals, Pennsylvania, USA) was added and incubated for 10 min in the dark, RT. To stop the reaction, 100 μL of 1 mM HCl was added, prior to reading on an ELISA plate reader at 450 nm [44 (link)].

Parry H.M., Zuo J., Frumento G., Mirajkar N., Inman C., Edwards E., Griffiths M., Pratt G, & Moss P. (2016). Cytomegalovirus viral load within blood increases markedly in healthy people over the age of 70 years. Immunity & Ageing : I & A, 13, 1.

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ELISA Analysis of Anti-Adenovirus Capsid Protein

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ELISA analysis of anti-Ad capsid protein sera was performed as previously described^{49 (link)}. Briefly, Ad capsid proteins were solubilized by vortexing of Ad-null in the presence of 0.1% triton-X solution for 10 min. Solubilized Ad capsid proteins were immobilized on a 96-well plate at a density of 5 × 10⁶ VP/well after 100-fold dilution by carbonate buffer. Anti-Ad capsid protein sera were 400-fold diluted by PBST containing Immunoblock, and were added to the well, followed by a 2-h incubation at 37 °C. The plates were then washed with PBST and incubated with biotin-labeled goat anti-mouse IgG antibody (SouthernBiotech, Birmingham, AL) for a 2-h incubation at 37 °C. After washing, streptavidin-HRP (SouthernBiotech) was added to the well, followed by a 1-h incubation at room temperature. Finally, TMB ELISA Peroxidase Substrate (Rockland Immunochemicals, Gilbertsville, PA) was added. The reaction was stopped by the addition of 0.5 mol/l HCl, and absorbance was read at 450 nm on a TriStar LB941 multimode reader (Berthold Technologies, Bad Wildbad, Germany).

Tomita K., Sakurai F., Iizuka S., Hemmi M., Wakabayashi K., Machitani M., Tachibana M., Katayama K., Kamada H, & Mizuguchi H. (2018). Antibodies against adenovirus fiber and penton base proteins inhibit adenovirus vector-mediated transduction in the liver following systemic administration. Scientific Reports, 8, 12315.

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IgM Antibody ELISA Protocol

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ELISA plates (M9410; Sigma) were coated with goat anti-mouse Ig (5300–05B; Southern Biotechnology) or NP-BSA overnight at 4°C and blocked with 3% nonfat dry milk for 2 h. A Standard curve of IgM antibody (5300–01B; Southern Biotechnology) and serum samples were incubated at 4°C overnight. The next day, the plates were washed and incubated with HRP-conjugated goat anti-mouse secondary antibody (1021–05; Southern Biotechnology) for 2 h at room temperature. The plates were developed using TMB ELISA peroxidase substrate (800–666-7625; Rockland) and the reaction was stopped using 0.2M sulfuric acid. Plates were read using ELx808 Absorbance plate reader (BioTek).

Kania A.K., Price M.J., George-Alexander L.E., Patterson D.G., Hicks S.L., Scharer C.D, & Boss J.M. (2022). H3K27me3 demethylase UTX restrains plasma cell formation. Journal of immunology (Baltimore, Md. : 1950), 208(8), 1873-1885.

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Quantifying Anti-Adenoviral Antibody Levels

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C57BL/6 mice were intravenously administered Ad vectors at 1 × 10¹⁰ IFU/mouse. Blood samples were collected via retro-orbital bleeding fourteen days after administration. Anti-Ad antibody levels in the serum were determined by enzyme-linked immunosorbent assay (ELISA). For the ELISA, a 96-well plate was coated with Ad-null (5 × 10⁵ IFU/well) overnight at 4 °C, washed with PBS-0.05% Tween (PBST), and blocked in ImmunoBlock (DS Pharma Biomedical, Osaka, Japan) for 1 hour at room temperature. The serum samples (diluted 1:500) were added to the antigen-coated plate and incubated for 2 hours at 37 °C. The plate was washed with PBST and incubated with biotin-conjugated goat anti-mouse IgG (H+L) (SouthernBiotech, Birmingham, AL) for 2 hours at 37 °C. The plates was then washed with PBST and incubated with streptavidin-HRP (SouthernBiotech) for 1 hour at room temperature. Finally, the plate was washed with PBST and TMB ELISA Peroxidase Substrate (Rockland Immunochemicals, Gilbertsville, PA) was added. The reaction was stopped by the addition of 0.5 mol/l HCl, and absorbance was read at 450 nm on a TriStar LB941 (Berthold Technologies, Bad Wildbad, Germany).

Shimizu K., Sakurai F., Tomita K., Nagamoto Y., Nakamura S.I., Katayama K., Tachibana M., Kawabata K, & Mizuguchi H. (2014). Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome. Molecular Therapy. Methods & Clinical Development, 1, 14035-.

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Quantitative Mouse IgM ELISA Assay

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Flat-bottom ELISA plates (Sigma M9410–1CS) were coated with goat anti-mouse Ig (Southern Biotechnology 5300–05B). Plates were incubated overnight at 4°C, washed (1X PBS with 0.05% Tween 20), blocked with 1% non-fat dry milk for two hours at room temperature, washed, incubated with diluted serum samples or mouse IgM (Southern Biotechnology 5300–01B) for two hours at room temperature, washed, incubated with 1:1000 diluted HRP-conjugated goat anti-mouse IgM (Southern Biotechnology 1021–05) for two hours at room temperature, and washed. Plates were incubated with TMB ELISA peroxidase substrate (Rockland TMBE-1000) for twenty minutes at room temperature (50 μl/well) to develop a color reaction. 50 μl of 0.2 M sulfuric acid was applied to each well to stop the HRP color reaction. After twenty minutes, optical density at 450 nm (OD₄₅₀) was collected by a Synergy HT Multi-Mode Microplate Reader (BioTek).

Haines R.R., Barwick B.G., Scharer C.D., Majumder P., Randall T.D, & Boss J.M. (2018). The histone demethylase LSD1 regulates B cell proliferation and plasmablast differentiation. Journal of immunology (Baltimore, Md. : 1950), 201(9), 2799-2811.

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IgM Antibody Secretion Quantification

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Equal numbers of DMSO- or GSK-J4-treated plasmablasts were cultured in fresh B cell media. After 2.5hr, the supernatant was collected and used to perform ELISA. ELISA plates (Sigma M9410) were coated with goat anti-mouse Ig (Southern Biotechnology 5300-05B) overnight at 4° C and blocked with 3% nonfat dry milk. Standard IgM antibody (Southern Biotechnology 5300-01B) and collected media supernatants were incubated for 2 hr at room temperature, followed by washes, and incubation with HRP-conjugated goat anti-mouse secondary antibody (Southern Biotechnology 1021-05) for 2 hr at room temperature. The plates were developed using the TMB ELISA peroxidase substrate (Rockland 800-666-7625) and the reaction was stopped using 0.2M sulfuric acid. Plates were read using a Synergy HT Multi-Mode Microplate Reader (BioTek).

Liang F.T, & Philipp M.T. (2000). Epitope Mapping of the Immunodominant Invariable Region of Borrelia burgdorferi VlsE in Three Host Species. Infection and Immunity, 68(4), 2349-2352.

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Determination of PE-specific Antibody Isotypes

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For determination of PE-specific IgM, IgA, IgG1, IgG2a, IgG2b, IgG2c, and IgG3 antibodies, flat-bottom ELISA plates (Evergreen Scientific) were coated with PE (Prozyme) diluted in borate-buffered saline to a concentration of 5 μg/ml. Plates were incubated overnight at 4 °C, washed, blocked with 3% non-fat dry milk for 2 h at room temperature, washed, and diluted sera samples were added. Following a second overnight incubation at 4 °C, plates were washed and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM, IgG1, IgG2b, IgG2c, and IgG3 (Southern Biotechnology) were applied. After a final wash, TMB ELISA peroxidase substrate (Rockland Immunochemicals) was applied to develop a color reaction, and optical density at 650 nm (OD₆₅₀) was collected by a Synergy HT Multi-Mode Microplate Reader (BioTek). The first three wash steps were performed with PBS containing 0.05% Tween 20 while the final wash was performed with PBS. Antibody concentrations were determined by comparing antibody levels for experimental samples with the standard curves of purified mouse IgM, IgA, IgG1, IgG2a, IgG2b, IgG2c, and IgG3 (Southern Biotech). Significant differences in antibody levels between samples were determined using two-sample t-tests with a P value of 0.05 being considered significant.

Barwick B.G., Scharer C.D., Martinez R.J., Price M.J., Wein A.N., Haines R.R., Bally A.P., Kohlmeier J.E, & Boss J.M. (2018). B cell activation and plasma cell differentiation are inhibited by de novo DNA methylation. Nature Communications, 9, 1900.

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Quantitative Biotinylated MUC16 ELISA

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96-well clear, flat-bottom plates (Thermo Scientific, 14-245-153) were coated with 1ug/ml BTM diluted in 0.1M sodium bicarbonate coating buffer (pH 8.0) overnight at 4C. Each well was washed with PBS-T (0.05% Tween-20) and subsequently blocked with PBS+2% BSA at room temperature for 1 hour. Wells were then washed with PBS-T before the addition of either biotinylated BiTEDs (Biotin labeling kit; Roche, 11418165001) or biotinylated MUC16^ecto-BiTEDs with free rhCA-125 (R&D systems, 5609-MU). Ratios of 1:1, 5:1, and 10:1 rhCA-125 to coated BTM were used. Following a one-hour incubation at room temperature on a plate rocker, wells were washed with PBS-T. Streptavidin-HRP A (R&D systems, 890803) at 1:200 in PBS was then added to each well and incubated for 1 hour at room temperature under foil. Each well was washed with PBS-T before the addition of TMB ELISA Peroxidase Substrate (Rockland) and allowed to develop for 20 minutes, protected from light, at room temperature. Reactions were then quenched with 0.6N sulfuric acid. Wavelengths of 450nm and 540nm (for plate refraction correction) were measured via SpectraMax iD3 Microplate Reader. VEGF detection was performed on OVCAR3 cells cultured in 6-well plates cultured for 48 hours in complete RPMI according to manufacturer’s instructions (R&D systems, DVE00).

Yeku O.O., Rao T.D., Laster I., Kononenko A., Purdon T.J., Wang P., Cui Z., Liu H., Brentjens R.J, & Spriggs D. (2021). Bispecific T-Cell Engaging Antibodies Against MUC16 Demonstrate Efficacy Against Ovarian Cancer in Monotherapy and in Combination With PD-1 and VEGF Inhibition. Frontiers in Immunology, 12, 663379.

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Influenza Antibody ELISA Assay

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ELISA plates (Evergreen Scientific, 52-333801301F) were coated with 5 μg/ml capture antibody (Southern Biotech, 5300-01B) or 1 μg/ml influenza hemagglutinin (HA) at 4°C overnight and blocked with 1% non-fat dry milk at room temperature for 2 hours. Standard antibodies and serum were bound to the plates at 4°C overnight, plates were washed, and secondary HRP-conjugated goat anti-mouse IgM or IgG antibodies were added for 2 hours at room temperature. Plates were developed with TMB ELISA peroxidase substrate (Rockland, #800-666-7625) and the reaction stopped with 0.2 M sulphuric acid. Plates were read at 450 nm with the Gen5 software.

Guo M., Price M.J., Patterson D.G., Barwick B.G., Haines R.R., Kania A.K., Bradley J.E., Randall T.D., Boss J.M, & Scharer C.D. (2017). EZH2 represses the B cell transcriptional program and regulates antibody secreting cell metabolism and antibody production. Journal of immunology (Baltimore, Md. : 1950), 200(3), 1039-1052.

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