Overview: Human Neutrophil Elastase (HNE) belongs to the chymotrypsin family of serine proteases able to solubilize fibrous elastin, an important extracellular matrix protein that has the unique property of elastic recoil, and plays a major structural function in lungs, arteries, skin and ligaments. HNE is primarily located in the azurophil granules of polymorphonuclear leucocytes, but has also been detected in nuclear membrane, Golgi complex, ER and the mitochondria of these cells. Promyelocytes and more differentiated myeloid cells are the only cells that contain the elastase. The enzyme is involved in the tissue destruction and inflammation and implicated in numerous disease, including emphysema, chronic obstructive pulmonary disease, cystic fibrosis, acute respiratory distress syndrome, ischemic-reperfusion injury, rheumatoid arthritis, and severe congenital neutropenia (1-3). For a long time elastase has been the object of extensive research to develop potent inhibitors that target its destructive and pro-inflammatory action. A sensitive and extremely selective assay to measure active HNE is essential to tracking enzyme involvement in such pathological processes.
References: 1 .Moraes, T.J., et al. 2003. Crit. Care Med.31, 189. 2. Horwitz, M., et al. 2003. Cancer Invest.21, 579. 3. Scaglioni, P.P. and Pandolfi, P.P. 2003. Nature426, 512.
Application: A unique and highly sensitive immunocapture/activity assay kit to analyze human neutrophil elastase (HNE) activity in cell lysates and body fluids. The assay utilizes a monoclonal antibody, specific for HNE, to first capture the enzyme from the crude sample. The remaining sample is washed away and the enzyme activity is assayed using a fluorogenic elastase substrate.
Assay Range: 1. 0.625-20 ng/ml for 4 hours assay 2. 0.0625-2 ng/ml for 24 hours assay
Kit Contents:
1.
Anti-HNE Coated Microtiter Plate: 1-96 well polystyrene microtiter plate (6 x 16-well strips) coated with a monoclonal antibody against human neutrophils elastase
2.
HNE Control: One vial, 80 ml of human neutrophil elastase (HNE, Cat. No. 368042) supplied at 5 mg/ml
3.
AMC Standard: One vial, 100 ml 2 mM 7-amino-4-methylcoumarin (AMC, Cat. No. 164545)) in DMSO
4.
Substrate: One vial, 60 ml of 100-fold concentrated MeOSuc-Ala-Ala-Pro-Val-AMC (Cat. No. 324740) in DMSO
5.
Assay Buffer: One bottle, 20 ml of Tris buffer, pH 7.5
6.
Sample Buffer: One bottle, 25 ml of 20-fold concentrated buffer with surfactant
7.
Sealer: Two each
Features
Benefits
• 96-well format • Combination immunocapture/activity assay • Fluorogenic substrate • Complete assay kit; includes antibody-coated plate, HNE standard, calibration standard, and necessary buffers for performing the activity assay
• High throughput for screening multiple samples at the same time • Confers specificity for HNE when using biological systems that otherwise contain a mixture of related enzymes • Allows for more sensitive assay • Convenient, easy to use; customer only needs to supply reagents for sample preparation
Assay Characteristics:
AMC Standard: Calibration curve generated with the free AMC standard included in the kit.
HNE Activity (Figure1): The activity of native HNE, included in the kit, using MeoSuc-Ala-Ala-Pro-Val AMC at pH 7.5 after 4-hour incubation at 37°C. Fluorescence reading: excitation 360 nm, emission 460 nm.
HNE Activity (Figure 2): The activity of native HNE, included in the kit, using MeoSuc-Ala-Ala-Pro-Val AMC at pH 7.5 after 24-hour incubation at 37°C. Fluorescence reading: excitation 360 nm, emission 460 nm.
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Overview: Human Neutrophil Elastase (HNE) belongs to the chymotrypsin family of serine proteases able to solubilize fibrous elastin, an important extracellular matrix protein that has the unique property of elastic recoil, and plays a major structural function in lungs, arteries, skin and ligaments. HNE is primarily located in the azurophil granules of polymorphonuclear leucocytes, but has also been detected in nuclear membrane, Golgi complex, ER and the mitochondria of these cells. Promyelocytes and more differentiated myeloid cells are the only cells that contain the elastase. The enzyme is involved in the tissue destruction and inflammation and implicated in numerous disease, including emphysema, chronic obstructive pulmonary disease, cystic fibrosis, acute respiratory distress syndrome, ischemic-reperfusion injury, rheumatoid arthritis, and severe congenital neutropenia (1-3). For a long time elastase has been the object of extensive research to develop potent inhibitors that target its destructive and pro-inflammatory action. A sensitive and extremely selective assay to measure active HNE is essential to tracking enzyme involvement in such pathological processes.
Add Innozyme Human Neutrophil Elastase Immunocapture Activity Assay KIt (Cat. No. 