Neutrophil elastase (NE) is a protein biomarker of infection and inflammation is shown to correlate with the severity of several respiratory diseases such as cystic fibrosis, COPD and bronchiectasis. This establishes NE as a frequently used COPD biomarker and CF biomarker for research.  NE has long been used as an endpoint in clinical studies aimed at evaluating the efficacy of new anti-elastase inhibitors (neutrophil elastase inhibitors) or other anti-inflammatory therapies.

For more about NE as a neutrophil serine proteases (NSP) and biomarker, read more here >>>>

 

Principle of the Assay:

The ProteaseTag® Active NE Immunoassay utilizes ProteaseTag® technology to specifically detect and quantify active NE.  The activity assay is a more specific alternative to a standard antigen or a total neutrophil elastase assay.

During a short incubation, the NE-Tag is coated onto the immunoassay plate before washing to remove excess.  Standards and samples are added, and the NE-Tag will interact with the active site of NE during a second incubation step. Active NE present in the solutions will irreversibly bind to the NE-Tag while latent and inhibitor bound NE will be removed by washing. A horseradish peroxidase (HRP) conjugated anti-NE antibody is added to each test well and incubated. This detection antibody attaches to bound NE with unbound antibody subsequently removed by washing. A color forming substrate containing TMB is added to each test well and reacts with HRP to generate a blue colored product. This enzymatic reaction is subsequently stopped by the addition of acidic stop solution to each test well (a yellow solution is formed). The color intensity (absorbance) is read at 450 nm using a plate reader.

Sufficient reagents are provided to allow for two standard curves

Freezer Box Components (stored at -20°C)

NE-Tag 1 vial containing 4 μl (concentration 10 mM) of capture probe in DMF
NE Standard 2 vials containing 6 μl of NE (concentration 100 μg/ml) in an acidic buffer

*Each vial can only be used once

NE Conjugate 1 vial containing 25 μl of anti-human neutrophil elastase antibody conjugated to HRP in a stability solution

 

Main Box Components (stored at 2-8°C)

Immunoassay Plate 96 well strep coated microtiter plate (pre-blocked)
NE Wash Buffer A Concentrate 24 ml of a 25-fold concentration of buffered surfactant with preservatives
NE Wash Buffer B Concentrate 8 ml of a 25-fold concentration of buffer with preservatives
NE Standard Diluent 15 ml of buffer with preservatives (ready to use)
NE Reagent Diluent 2 x 13 ml of buffered surfactant with preservatives (ready to use)
TMB Substrate 12 mL of tetramethylbenzidine solution
Stop Solution 6 mL of 2N sulfuric acid
Plate Sealers 4 adhesive strips

Format:           96 well

Species:           Human

Sample:           Sputum, bronchial alveolar lavage fluid (BALF), wound exudate

Range:             15.625 – 1000 ng/ml

LOD:                7.2 ng/ml

Specificity:       Only detects active NE

Run Time:        130 min of incubation

Cross Reactivity:  reactive with other species, but no official testing results provided

Standard Source: native protein