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| Product Name | Myeloperoxidase (MPO) Activity Colorimetric Assay Kit |
| Catalog No. | ETR-HMM-0064 |
| Description | A colorimetric assay for measuring myeloperoxidase enzyme activity in biological samples. MPO, primarily expressed in neutrophils and macrophages, catalyzes the oxidation of halide ions by hydrogen peroxide to produce hypohalous acids; the assay uses the chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine) to measure H2O2-dependent MPO activity at 460 nm. |
| Intended Use | Quantitative measurement of MPO activity as a biomarker of neutrophil infiltration and activity in inflammation research, atherosclerosis, acute lung injury, inflammatory bowel disease, and assessment of antibacterial host defense mechanisms. |
| Principle / Technology | MPO (EC 1.11.1.7) uses hydrogen peroxide as an oxidant to catalyze the two-electron oxidation of TMB substrate; the resulting oxidized TMB cation radical product is a blue-green chromophore absorbing at 450–460 nm; the TMB oxidation rate is directly proportional to MPO activity; in the more sensitive alkaline phosphatase amplification format, MPO activity is measured through a coupled amplification reaction. |
| Detection Method | Colorimetric absorbance measurement at 460 nm; kinetic or endpoint measurement; also detectable fluorometrically in adapted protocols |
| Sample Type | Tissue homogenates from inflammatory sites (lung, colon, joint, skin), activated neutrophil lysates, differentiated HL-60 cell lysates, bronchoalveolar lavage fluid, serum and plasma (at high neutrophil burden) |
| Performance Range / Specifications | MPO activity range: 0.01–100 mU/mL; tissue MPO content in inflamed versus non-inflamed mouse colon: 2–100 mU/mg protein; one unit of MPO activity = amount oxidizing 1 μmol H2O2 per minute at pH 7.0 at 25°C |
| Sensitivity / LOD | Lower detection limit: approximately 0.1 mU/mL using the standard colorimetric format; sample input as little as 50 mg tissue homogenate or 5 × 10^5 activated neutrophils per assay |
| Specificity | TMB oxidation rate by MPO is H2O2-dependent and requires the native MPO heme moiety; the assay is inhibited by sodium azide, cyanide, and aminotriazole confirming heme-dependent peroxidase mechanism; eosinophil peroxidase (EPO) can also oxidize TMB with similar rate; differentiation from EPO requires selective inhibitors |
| Reaction Conditions / Protocol | Prepare tissue homogenates in lysis buffer (50 mM potassium phosphate pH 7.4 + 0.5% HTAB detergent, 3 freeze-thaw cycles for complete cell lysis); centrifuge 10,000 × g 10 minutes at 4°C; prepare serial dilutions of supernatant; add 90 μL TMB Substrate Reagent per well; add 10 μL sample; incubate at room temperature for 2–5 minutes protected from light; add 100 μL Stop Solution (H2SO4); measure absorbance at 460 nm; calculate MPO activity from MPO standard curve or positive control comparison |
| Components / Formulation | TMB Substrate Reagent (optimized TMB in citrate-acetate buffer with H2O2), Stop Solution (0.2 M H2SO4), MPO Lysis Buffer (potassium phosphate with HTAB), MPO Standard (purified human MPO, lyophilized, certified activity units), Assay Diluent |
| Storage Conditions | All reagents at 2–8°C protected from light; MPO standard at -20°C in single-use aliquots, stable 12 months; TMB substrate sensitive to light, use within 30 days of opening |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 96 assays, 192 assays, 384 assays (96-well format) |
| Product Form | Liquid substrate and buffer components with lyophilized MPO standard |
| Quality Control | Each lot validated with purified human MPO standard; linearity confirmed (R2 ≥0.98) across the activity range; inhibition by 1 mM sodium azide >90% confirms heme-dependent mechanism; lot-to-lot MPO activity within 20% of reference value |
| Key Features | Certified purified human MPO standard for activity comparison across labs; HTAB detergent in lysis buffer efficiently extracts granule-bound MPO from neutrophils; colorimetric detection suitable for tissue samples where protein-containing matrix would interfere with more sensitive fluorometric methods |
For research use only, not for clinical use.
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