O phenylenediamine dihydrochloride peroxidase substrate

Manufactured by Merck Group

Sourced in United States

O-phenylenediamine dihydrochloride peroxidase substrate is a chemical compound used as a colorimetric detection reagent in various laboratory applications. It serves as a substrate for the enzyme peroxidase, which catalyzes the oxidation of the compound, resulting in a colored product that can be detected and quantified spectrophotometrically.

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Lab products found in correlation

Goat anti human igg hrp, by Jackson ImmunoResearch (3 mentions) Maxisorp, by Thermo Fisher Scientific (3 mentions) Prism v8, by GraphPad (2 mentions) Baculovirus expression system, by Sino Biological (1 mentions) Hrp conjugated goat anti human igg, by Southern Biotech (1 mentions) Peroxidase substrate, by Merck Group (1 mentions) Horseradish peroxidase conjugated streptavidin, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions)

6 protocols using o phenylenediamine dihydrochloride peroxidase substrate

Binding Curves of Chimeric and Humanized SAP Antibodies

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Example 6

1 μg/mL human SAP was directly immobilised onto an ELISA plate and blocked with 1% BSA/TBS plus 0.05% TWEEN20. Anti-SAP antibodies from the test supernatants or purified material were titrated across the plate. Bound antibody was detected by treatment with goat anti-human Kappa Light Chains peroxidase conjugate (Sigma, A7164). The ELISA was developed using O-phenylenediamine dihydrochloride (OPD) peroxidase substrate (Sigma, P9187).

FIG. 3 shows the binding curves for chimeric antibodies cSAP-E and cSAP-K. The profile of the curves for the chimeric antibodies is the same as that of the equivalent hybridomas.

FIG. 4 shows the binding curves for SAP-K H0L0, SAP-K H1L0, SAP-K H2L0 and SAP-K H3L0 compared to the SAP-K chimera and the SAP-E H1L1 compared to the SAP-E chimera. An irrelevant human IgG1 kappa antibody was also tested as a negative control. The data shows that humanisation of the SAP-K antibody resulted in a loss of human SAP binding activity of approximately 2-fold compared to the parental SAP-K chimera, whilst the humanised SAP-E antibody retained binding activity compared to the parental SAP-E chimera.

US10221234B2. Antigen binding proteins (2019-03-05). GLAXO GROUP LIMITED [GB]. Inventors: Tejinder Kaur Bhinder [GB], Susannah Karen Ford [GB], Volker Germaschewski [GB], Alan Peter Lewis [GB], Mark Brian Pepys [GB].

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Anti-Vimentin Antibody ELISA Assay

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Anti-vimentin antibodies were measured by a standard ELISA against recombinant human vimentin purified from a baculovirus expression system (SinoBiological, Wayne, PA, USA). Vimentin in bicarbonate buffer (5 μg/mL), pH 9.4, was coated on ELISA plates overnight at 4 °C. All sera were tested at a 1:100 dilution. Serial dilutions of a pooled serum sample were used as a calibrator for each plate. Bound antibody was detected with HRP-conjugated goat anti-human IgG (SouthernBiotech, Birmingham AL, USA), and the enzyme activity was determined with an o-phenylenediamine dihydrochloride (OPD) peroxidase substrate (Sigma-Aldrich, St. Louis, MO, USA). The reaction was stopped with 2.5 N sulfuric acid. Results are presented as absorbance at 490 nm. The mean O.D. +2SD of the control group was used as a cut-off to determine the prevalence of anti-vimentin antibodies.

Bagavant H., Cizio K., Araszkiewicz A.M., Papinska J.A., Garman L., Li C., Pezant N., Drake W.P., Montgomery C.G, & Deshmukh U.S. (2022). Systemic immune response to vimentin and granuloma formation in a model of pulmonary sarcoidosis. Journal of Translational Autoimmunity, 5, 100153.

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SARS-CoV-2 Spike Protein Binding Assay

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Assays were performed in 96-well plates (MaxiSorp; Thermo) coated with 100 μL of recombinant S from WA1/2020, B.1.351, B.1.617.2, and BA.1 strains of SARS-CoV-2, N-terminal domain of BA.1, receptor binding domain of BA.1, or S2 domain of WA1/2020, or bovine serum albumin diluted to 1 μg/mL in PBS, and plates were incubated at 4°C overnight. Plates then were blocked with 10% FBS and 0.05% Tween 20 in PBS. Purified mAbs were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween 20 in PBS. Goat anti-human IgG-HRP (goat polyclonal, Jackson ImmunoResearch, 1:2,500) was diluted in blocking buffer before adding to plates and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween 20 in PBS and 3 times with PBS before the addition of o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich). Reactions were stopped by the addition of 1 M hydrochloric acid. Optical density measurements were taken at 490 nm.

Alsoussi W.B., Malladi S.K., Zhou J.Q., Liu Z., Ying B., Kim W., Schmitz A.J., Lei T., Horvath S.C., Sturtz A.J., McIntire K.M., Evavold B., Han F., Scheaffer S.M., Fox I.F., Parra-Rodriguez L., Nachbagauer R., Nestorova B., Chalkias S., Farnsworth C.W., Klebert M.K., Pusic I., Strnad B.S., Middleton W.D., Teefey S.A., Whelan S.P., Diamond M.S., Paris R., O’Halloran J.A., Presti R.M., Turner J.S, & Ellebedy A.H. (2022). SARS-CoV-2 Omicron boosting induces de novo B cell response in humans. bioRxiv.

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SARS-CoV-2 Antibody Binding Assay

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Assays were performed in 96-well plates (MaxiSorp; Thermo) coated with 100 μl of recombinant S, RBD, N-terminal domain of S (SinoBiological), OC43 S, HKU1 S or bovine serum albumin diluted to 1 μg ml⁻¹ in PBS, and plates were incubated at 4 °C overnight. Plates then were blocked with 10% FBS and 0.05% Tween 20 in PBS. Plasma or purified monoclonal antibodies were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween 20 in PBS. Goat anti-human IgG-HRP (goat polyclonal, Jackson ImmunoResearch, 1:2,500), IgA (goat polyclonal, Jackson ImmuoResearch, 1:2,500) or IgM (goat polyclonal, Caltag, 1:4,000) were diluted in blocking buffer before adding to wells and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween 20 in PBS and 3 times with PBS before the addition of o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich). Reactions were stopped by the addition of 1 M hydrochloric acid. Optical density measurements were taken at 490 nm. The area under the curve for each monoclonal antibody and half-maximal binding dilution for each plasma sample were calculated using Graphpad Prism v.8.

Turner J.S., O’Halloran J.A., Kalaidina E., Kim W., Schmitz A.J., Zhou J.Q., Lei T., Thapa M., Chen R.E., Case J.B., Amanat F., Rauseo A.M., Haile A., Xie X., Klebert M.K., Suessen T., Middleton W.D., Shi P.Y., Krammer F., Teefey S.A., Diamond M.S., Presti R.M, & Ellebedy A.H. (2021). SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses. Nature, 596(7870), 109-113.

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Amyloid-β and HMGB1 Binding Assay

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Ig-fusion protein of sRAGE in a concentration of 5 mg/mL was bound to protein A (1 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) coated wells (Maxisorb ELISA plates, Sigma-Aldrich). Unbound proteins were washed away, and the wells were blocked with BSA (Sigma-Aldrich). Biotinylated amyloid β 1–42 peptide (20 nM, American Peptide, Sunnyvale, CA, USA) or biotinylated HMGB1 (20 nM) were bound to the wells in the presence or absence of inhibitors, and unbound ligands were washed away. Bound biotinylated ligands were detected with horse radish peroxidase conjugated streptavidin (Sigma-Aldrich) and peroxidase substrate (Sigma-Aldrich). The color was developed using o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich). Absorbance at 490 nm was measured.

Rouhiainen A., Nykänen N.P., Kuja-Panula J., Vanttola P., Huttunen H.J, & Rauvala H. (2018). Inhibition of Homophilic Interactions and Ligand Binding of the Receptor for Advanced Glycation End Products by Heparin and Heparin-Related Carbohydrate Structures. Medicines, 5(3), 79.

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SARS-CoV-2 Antibody Binding Assay

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Assays were performed in 96-well plates (MaxiSorp, Thermo Fisher Scientific) coated with 100 μl of Flucelvax 2019/2020 or recombinant S in PBS, and plates were incubated at 4 °C overnight. Plates were then blocked with 10% FBS and 0.05% Tween-20 in PBS. Serum or plasma were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween-20 in PBS. Goat anti-human IgG-HRP ( Jackson ImmunoResearch, 1:2,500) was diluted in blocking buffer before adding to wells and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween-20 in PBS, and then washed 3 times with PBS before the addition of o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich). Reactions were stopped by the addition of 1 M HCl. Optical density measurements were taken at 490 nm. The half-maximal binding dilution for each serum or plasma sample was calculated using nonlinear regression (GraphPad Prism v.8). The limit of detection was defined as 1:30.

Turner J.S., Kim W., Kalaidina E., Goss C.W., Rauseo A.M., Schmitz A.J., Hansen L., Haile A., Klebert M.K., Pusic I., O'Halloran J.A., Presti R.M, & Ellebedy A.H. (2021). SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans. Nature, 595(7867).

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