The sputum supernatant EDN was quantified using enzyme-linked immunosorbent assay kits (Medical and Biological Laboratory Co., Ltd., Nagoya, Japan), whereas the sputum supernatant NE was measured using an NE activity assay kit according to the manufacturer’s instructions (Cayman Chemicals, Ann Arbor, MI, USA). Measurements were performed in duplicate. The lower limit of detection for the EDN assay was 0.62 ng/mL.
Ne activity assay kit
Manufactured by Cayman Chemical
Sourced in Japan, United States
The NE activity assay kit is a laboratory tool designed to measure the enzymatic activity of neutrophil elastase (NE). It provides a quantitative assessment of NE levels within a sample. The kit contains the necessary reagents and protocols to perform the assay.
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3 protocols using ne activity assay kit
1
Quantifying Sputum EDN and NE
2
Sputum MMP-9 and Neutrophil Elastase Assay
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Sputum matrix metalloproteinase (MMP)-9 level was measured using Luminex multiplex assay (Bio-Rad, USA) [13 (link)] with the Bio-Plex Reader (Bio-Rad). The lower and upper bounds of detection were 0.14 and 302.50 ng·mL−1, respectively, for MMP-9. Free neutrophil elastase (NE) activity was quantified in sputum supernatant using an NE activity assay kit according to the manufacturer's instructions (Cayman Chemicals, USA). Measurements were performed in duplicate and the mean was included for further analysis.
3
Quantifying Neutrophil Elastase Activity
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Enzymatic activity of NE was evaluated using the NE activity assay kit (Cat#600610; Cayman Chemical; Ann Arbor, MI) as previously described (28 (link)). Neutrophil elastase at 18 mU/ml was used to make a standard curve ranging from 0.156 mU/ml to 10 mU/ml. Standards and samples were added to a black, flat-bottom, 96-well plate. Ten μl of substrate solution consisting of Cell-Based Assay DMF and (Z-Ala-Ala-Ala-Ala)2Rh110 was added to each standard and sample, which is selectively cleaved by elastase to yield the highly fluorescent compound R110. Fluorescence was analyzed with an excitation wavelength of 485 nm and an emission wavelength of 525 nm using a microplate fluorimeter (Varioskan Flash, Thermo Scientific). Kinetic fluorescent measurements were taken every 2 minutes for 2 hours at 37°C. Average fluorescence was taken for each well and quantified in mU/ml with the help of the NE standard.
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