Chromogenic assay

Manufactured by Siemens

Sourced in Germany

Chromogenic assay is a laboratory technique used to measure the concentration or activity of a target analyte in a sample. It relies on a color-producing chemical reaction that occurs when the target analyte interacts with specific reagents. The intensity of the color produced is proportional to the amount of the target analyte present, which can be quantified using spectrophotometric measurements.

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Lab products found in correlation

Zymutest activatable tafi, by Hyphen Biomed (1 mentions) Pai 1 antigen, by Hyphen Biomed (1 mentions) Thromborel s, by Siemens (1 mentions) Multifibren u, by Siemens (1 mentions) P selectin, by R&D Systems (1 mentions) 1450 microbeta, by PerkinElmer (1 mentions) Concanavalin a (cona), by Merck Group (1 mentions) Naocl, by Merck Group (1 mentions) Helixate nexgen, by CSL Behring (1 mentions)

5 protocols using chromogenic assay

Comprehensive Cardiovascular Biomarker Evaluation

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Blood samples were drawn from antecubital veins, with minimal stasis. The complete blood cell count, lipid profile, glucose, and creatinine were assayed by routine laboratory techniques. High-sensitivity C-reactive protein (hsCRP) and Lp(a) were determined using immunoturbidimetry (Roche Diagnostics, Mannheim, Germany). Fibrinogen was measured using the Clauss method (Multifibren U, Siemens Healthcare, Marburg, Germany). Factor II activity was determined using a coagulometric method (Thromborel S, Siemens Healthcare). Plasma α2-antiplasmin and plasminogen were measured by chromogenic assays (Siemens Healthcare). Commercially available ELISA tests were used to determine TF antigen (Zymutest Total Tissue Factor Antigen), PAI-1 Antigen (Zymutest PAI-1 Antigen), and thrombin activatable fibrinolysis inhibitor (TAFI) zymogen (Zymutest Activatable TAFI, all Hyphen BioMed, Neuville-Sur-Oise, France).
To evaluate fibrin clot properties and thrombin generation, blood samples were mixed with 3.2% sodium citrate (vol/vol, 9:1) and centrifuged at 2000 × g for 10 min within 30 min since the draw. The supernatant was aliquoted and stored at −80 °C until further analysis. All measurements were performed by technicians who were blind to the origin of the samples.

Siudut J., Natorska J., Wypasek E., Wiewiórka Ł., Ostrowska-Kaim E., Wiśniowska-Śmiałek S., Plens K., Legutko J, & Undas A. (2020). Impaired Fibrinolysis in Patients with Isolated Aortic Stenosis is Associated with Enhanced Oxidative Stress. Journal of Clinical Medicine, 9(6), 2002.

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Plasma Biomarker Quantification Protocol

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To obtain citrated plasma, blood samples were mixed with 3.2% sodium citrate (9:1), centrifuged for 20 min and stored at − 80 °C. Fibrinogen was determined with the von Clauss method. Plasminogen and α2-antiplasmin were measured by chromogenic assays (Siemens, Munich, Germany). Commercially available immunoenzymatic assays were used to measure plasma PAI-1 antigen (Hyphen, Neuville-sur-Oise, France), TAFI (Hyphen, Neuville-sur-Oise, France), thrombomodulin (Diagnostica Stago, Parsippany, NJ, USA), P-selectin and platelet factor 4 (R&D Systems, Minneapolis, MN, USA).

Bryk A.H., Prior S.M., Plens K., Konieczynska M., Hohendorff J., Malecki M.T., Butenas S, & Undas A. (2019). Predictors of neutrophil extracellular traps markers in type 2 diabetes mellitus: associations with a prothrombotic state and hypofibrinolysis. Cardiovascular Diabetology, 18, 49.

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Measuring Anti-FVIII Antibodies and Inhibition in Mice

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Anti-FVIII IgG in mouse serum were measured by enzyme-linked immunosorbent assay (ELISA) and FVIII inhibitory titers were measured with a chromogenic assay (Siemens, Marburg, Germany).^{21 (link)} Proliferation of splenocytes was assessed in 96-well plates (0.25x10⁶ cells/well) with concanavalin A (Sigma) or BDD-FVIII for 72 h.^{22 (link)} Cell proliferation was measured by incorporation of [^{3 (link)}H]-thymidine (0.5 μCi/well) for an additional 18 h, using a β–counter (Microbeta 1450, Perkin Elmer).

Delignat S., Russick J., Gangadharan B., Rayes J., Ing M., Voorberg J., Kaveri S.V, & Lacroix-Desmazes S. (2019). Prevention of the anti-factor VIII memory B-cell response by inhibition of Bruton tyrosine kinase in experimental hemophilia A. Haematologica, 104(5), 1046-1054.

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Measuring FXIII Activity in Murine Plasma

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Murine plasma samples for FXIII activity measurement were collected from the retro-orbital venous sinus. FXIII activity in mouse plasma was measured using a chromogenic assay (Siemens Healthcare Diagnostics, Germany) following the manufacturer’s instructions. Results are expressed as a percentage of FXIII activity relative to a cohort of unchallenged sex and age matched WT mice from the same colony. Note that the FXIII activity assay does not distinguish between human- or murine-derived FXIII activity. Human rFXIII antigen in mouse plasma was measured by an enzyme immunoassay (Technozym EIA, Technoclone, Vienna) specific for human FXIII that detects both the FXIII-A₂B₂ complex and free A₂ dimer, but does not detect murine FXIII-A or-B. The rFXIII was used as reference material for calibration and control samples.

Andersson C., Kvist P.H., McElhinney K., Baylis R., Gram L.K., Pelzer H., Lauritzen B., Holm T.L., Hogan S., Wu D., Turpin B., Miller W, & Palumbo J.S. (2015). Factor XIII Transglutaminase Supports the Resolution of Mucosal Damage in Experimental Colitis. PLoS ONE, 10(6), e0128113.

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Oxidative Modification of FVIII Protein

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FVIII (Helixate NexGen, CSL-Behring, Marburg, Germany) was dialyzed against HEPES buffer (150 mM NaCl, 10 mM HEPES, 2.5 mM CaCl 2 pH 7.4) for 2 hr at 4°C. FVIII was mixed with NaOCl (Sigma-Aldrich, Darmstadt, Germany) at various molar ratios and incubated at 4°C for 30 min. The NaOCl concentration was determined by absorbance measurement at 292 nm using a molar extinction coefficient of 350 M -1 cm -1 . FVIII was then dialyzed against HEPES buffer for an additional 2 hr and concentration was determined by optical density at 280 nm. FVIII activity (FVIII:C) was determined using a chromogenic assay (Siemens, Marburg, Germany) using human plasma as standard. Loss of free amines was measured by fluorescamine fluorescence [13] . Briefly, 20 µl of native or HOCl-treated FVIII (1 µM) was added to 730 µl of borate buffer (200 mM, pH 8.5) and mixed while 250 µl of fluorescamine (20 µM) was added with vortexing. Following incubation at 25°C for 15 min, fluorescence was measured between 420 and 600 nm using an excitation wavelength of 390 nm. Carbonyl groups were quantified using a commercial ELISA kit (OxySelect Carbonyl ELISA).

Peyron I., Dimitrov J.D., Delignat S., Gangadharan B., Srivastava A., Kaveri S.V, & Lacroix-Desmazes S. (2018). Oxidation of factor VIII increases its immunogenicity in mice with severe hemophilia A. Cellular immunology, 325.

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