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CBA016 InnoZyme™ Human Neutrophil Elastase Immunocapture Activity Assay Kit

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Synonyme: HNE Immunocapture Activity Assay Kit

CBA016
  
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      Übersicht

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric
      Description
      OverviewA sensitive and selective assay for the measurement of human neutrophil elastase (HNE), a serine protease with the ability to solubilize fibrous elastin. Anti-HNE is immobilized onto a 96-well plate; activity is measured with a fluorogenic substrate (Ex: 360-380 nm; Em.: 440-460 nm).
      Catalogue NumberCBA016
      Brand Family Calbiochem®
      SynonymsHNE Immunocapture Activity Assay Kit
      Application Data
      Calibration curve generated with the free AMC standard included in the kit.

      The activity of native HNE, included in the kit, using MeoSuc-Ala-Ala-Pro-Val AMC at pH 7.5 after 4 h incubation at 37°C. Fluorescence reading: excitation 360 nm, emission 440-460 nm.

      The activity of native HNE, included in the kit, using MeoSuc-Ala-Ala-Pro-Val AMC at pH 7.5 after 24 h incubation at 37°C. Fluorescence reading: excitation 360 nm, emission 460 nm.

      Representation of HNE activity in human plasma assayed with Cat. No. CBA016. Fluorescence reading: excitation 360 nm, emission 460 nm.
      Materials Required but Not Delivered• Pipetman or multi-channel pipetman; use only pipettes that are carefully calibrated to their target volume
      • 37°C incubator
      • Microplate reader capable of measuring fluorescence at wavelengths 360-380 nm for excitation and 440-460 nm for emission
      • Ice bucket to keep reagents cold until use
      References
      Product Information
      Detection methodFluorometric
      Form96 Tests
      Format96-well plate
      Kit containsAnti-HNE-Coated 96-Well Plate, HNE Control, Substrate, AMC Standard, Assay Buffer, Sample Buffer, Plate Sealer, and a user protocol.
      Positive controlHuman neutrophil elastase
      Applications
      Biological Information
      Assay range0.625-20 ng/ml (4 h) and 62.5-2000 pg/ml (24 h)
      Assay time6 h
      Sample TypeCell lysates, body fluids, for screening HNE inhibitors
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe InnoZyme™ Human Neutrophil Elastase Immunocapture Activity Assay Kit is designed to measure human neutrophil elastase activity in cell lysates and body fluids, and for the screening of the enzyme inhibitors.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage -20°C
      Storage ConditionsThe unopened kit should be stored at -20°C. All kit components, once opened, can be stored for up to three months at the following conditions: assay buffer, AMC standard, substrate, sample buffer, and microwell plate at 4°C, and the HNE control at -20°C. Note: Once opened the HNE should be stored at -20°C in small aliquots to avoid freeze/thaw cycles.
      Protect from Light Protect from light
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsAnti-HNE-Coated 96-Well Plate, HNE Control, Substrate, AMC Standard, Assay Buffer, Sample Buffer, Plate Sealer, and a user protocol.
      Specifications
      Global Trade Item Number
      Bestellnummer GTIN
      CBA016 0

      Documentation

      InnoZyme™ Human Neutrophil Elastase Immunocapture Activity Assay Kit Analysenzertifikate

      TitelChargennummer
      CBA016
      Anwenderprotokoll

      Revision17-July-2008 JSW
      SynonymsHNE Immunocapture Activity Assay Kit
      Form96 Tests
      Format96-well plate
      Detection methodFluorometric
      StorageThe unopened kit should be stored at -20°C. All kit components, once opened, can be stored for up to three months at the following conditions: assay buffer, AMC standard, substrate, sample buffer, and microwell plate at 4°C, and the HNE control at -20°C. Note: Once opened the HNE should be stored at -20°C in small aliquots to avoid freeze/thaw cycles.
      Intended useThe InnoZyme™ Human Neutrophil Elastase Immunocapture Activity Assay Kit is designed to measure human neutrophil elastase activity in cell lysates and body fluids, and for the screening of the enzyme inhibitors.
      BackgroundHuman 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. 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.
      Principles of the assayThe InnoZyme™ HNE Activity Assay is a sensitive and selective assay to measure active human neutrophil elastase. The assay uses a monoclonal antibody to human NE immobilized on a 96-well microplate to specifically bind human neutrophil elastase. Activity of captured HNE is detected with the fluorogenic substrate MeOSuc-Ala-Ala-Pro-Val AMC. Released free AMC is measured fluorometrically at excitation 360-380 nm and emission 440-460 nm. Free AMC and HNE are provided for assay standardization. Enzyme detection ranges from 0.625-20 ng/ml for a 4-h incubation period at 37°C or 62.5-2000 pg/ml with a prolonged 24-h incubation.
      Materials provided• Anti-HNE Coated 96-Well Plate (Kit Component No. JA7890-1EA): 1 96-well polystyrene microplate (6 strips of 16 wells) coated with a monoclonal antibody against human neutrophil elastase
      • HNE Control (Kit Component No. JA7892-1EA): 1 vial, 80 µl of human neutrophil elastase supplied at 5 µg/ml
      • AMC Standard (Kit Component No. JA7742-100UL): 1 vial, 100 µl 2 mM 7-amino-4-methylcoumarin (AMC) in DMSO
      • Substrate (Kit Component No. JA7891-60UL): 1 vial, 60 µl of 100-fold concentrated MeOSuc-Ala-Ala-Pro-Val AMC in DMSO
      • Assay Buffer (Kit Component No. JA7893-20ML): 1 bottle, 20 ml of Tris buffer, pH 7.5
      • Sample Buffer (Kit Component No. JA7894-25ML): 1 bottle, 25 ml of 20-fold concentrated buffer with surfactant
      • Plate Sealer: 2 each
      Materials Required but not provided• Pipetman or multi-channel pipetman; use only pipettes that are carefully calibrated to their target volume
      • 37°C incubator
      • Microplate reader capable of measuring fluorescence at wavelengths 360-380 nm for excitation and 440-460 nm for emission
      • Ice bucket to keep reagents cold until use
      Reagent preparationNote: All reagents necessary to perform the assay are supplied with this kit. Bring all reagents except HNE control (keep this on ice) to 15-25°C before use. • Sample Buffer: Dilute the 20X Sample Buffer to make a 1X with dH2O. • Substrate Working Solution: Dilute MeOSuc-Ala-Ala-Pro-Val AMC substrate 100 times with assay buffer. • AMC Standard: The free AMC standard is supplied as a 2 mM stock solution. To create a calibration curve with the AMC standard a 0.625-20 µM concentration range is suggested for the fluorescence reading with excitation 360 nm and emission 460 nm. Example: Label 6 eppendorf tubes. Pipet 250 µl of assay buffer into the first tube and 125 µl into the remaining tubes. Add 2.5 µl of the 2 mM AMC standard to the first tube. Vortex and transfer 125 µl from this tube to the next tube. Continue serial dilutions by transferring 125 µl up to tube #6. • Human Neutrophil Elastase (HNE) Control: HNE is supplied as a 5 µg/ml stock solution. The following solutions are suggested for 4 and 24 h incubations: • 10 ng/ml HNE control, 4 h incubation: Pipet 998 µl of sample buffer into a tube. Add 2 µl of the 5 µg/ml HNE stock solution. Vortex for 2-3 s. • 1 ng/ml HNE control, 24 h incubation: Use the 10 ng/ml solution to prepare 1 ng/ml HNE control dilution. Pipet 450 µl of sample buffer into the tube and add 50 µl of the 10 ng/ml HNE solution. Vortex for 2-3 s.
      Detailed protocolNote: Bring all reagents, except HNE control (keep this on ice), and samples to room temperature before use. It is recommended that all samples and standards be assayed in duplicate.

      Note: The following procedure describes the assay calibrated with free AMC. For the experiment without AMC standard omit steps 4, 6, and 9.

      1. Prepare dilutions of control and samples.
      2. Remove the plate from the foil pouch.
      3. Pipet 100 µl of control or samples into each well. Blank should contain 100 µl of 1X sample buffer.
      4. Add 100 µl of 1X sample buffer to wells designated for AMC standard.
      5. Cover the plate with sealer and incubate for 1 h at room temperature with gently shaking.
      6. Prepare dilutions of AMC standard with assay buffer.
      7. Prepare 1X substrate by diluting 100X substrate stock solution with assay buffer.
      8. After the 1 h incubation wash the plate four times with 1X sample buffer. Remove the contents of the wells by inverting the plate over the sink and tapping it on paper towels.
      9. Pipet 50 µl of AMC standard solutions into the designated wells.
      10. Add 50 µl of assay buffer to wells containing controls, blanks, and samples.
      11. Add 50 µl of diluted 1X substrate to each well. Seal the plate well with adhesive wrap.
      12. Incubate at 37°C for 4 h (0.625-20 ng/ml HNE assay range detection) or 24 h (0.0625-2.0 ng/ml HNE assay range detection) in a humidified incubator.

      Note: All wells should be tightly closed during incubation at 37°C. Any change in liquid volume, due to evaporation, will influence fluorescence reading.

      13. Read the fluorescence at excitation 360-380 nm and emission 440-460 nm.
      CalculationsFor the assay with AMC calibration: It is recommended that the data be handled by a software package utilizing a linear regression curve-fitting program. If a software package is not available, construct a standard curve by plotting the mean fluorescence units for each standard on the Y-axis versus the corresponding concentration of AMC on the X-axis and draw a best-fit curve through the points on the graph. The HNE activity of the unknown samples can be interpolated from the standard curve and displayed as the amount of free AMC released per mg of protein per time. For the assay without AMC calibration: Activity of the enzyme is displayed in fluorescence units after subtracting the value of the blank, then calculating the mean fluorescence value for each sample in duplicate.
      Assay characteristics and examples

      Figure 1: AMC Standard

      Calibration curve generated with the free AMC standard included in the kit.


      Figure 2: HNE Activity

      The activity of native HNE, included in the kit, using MeoSuc-Ala-Ala-Pro-Val AMC at pH 7.5 after 4 h incubation at 37°C. Fluorescence reading: excitation 360 nm, emission 440-460 nm.


      Figure 3: HNE Activity

      The activity of native HNE, included in the kit, using MeoSuc-Ala-Ala-Pro-Val AMC at pH 7.5 after 24 h incubation at 37°C. Fluorescence reading: excitation 360 nm, emission 460 nm.


      Table 1: HNE Activity in Human Plasma

      Representation of HNE activity in human plasma assayed with Cat. No. CBA016. Fluorescence reading: excitation 360 nm, emission 460 nm.
      Assay Range0.625-20 ng/ml (4 h) and 62.5-2000 pg/ml (24 h)
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ and InnoZyme™ are trademarks of EMD Chemicals, Inc.