Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling.
BACKGROUND
During the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin.


METHODS
Monoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40).


RESULTS
Analyte recovery of EL-NE for human serum was between 85% and 104%, the analyte was stable for four freeze/thaw cycles and after 24 h storage at 4°C. EL-NE was specific for NE-degraded elastin. Levels of NE-generated elastin fragments for elastin incubated in the presence of NE were 900% to 4700% higher than those seen with CatG or MMP incubation or in intact elastin. Serum levels of NE-generated elastin fragments were significantly increased in patients with IPF (137%, p = 0.002) and in patients with lung cancer (510%, p < 0.001) compared with age- and sex-matched controls.


CONCLUSIONS
The EL-NE assay was specific for NE-degraded elastin. The EL-NE assay was able to specifically quantify NE-degraded elastin in serum. Serum levels of NE-degraded elastin might be used to detect excessive lung tissue degradation in lung cancer and IPF.
Nordic Bioscience A/S, Herlev, Denmark. jhk@nordicbioscience.com.|Department of Systems Biology, The Technical University of Denmark, Kgs. Lyngby, Denmark. jhk@nordicbioscience.com.
Jacob H
JH
Kristensen
Nordic Bioscience A/S, Herlev, Denmark. mk@nordicbioscience.com.
Morten A
MA
Karsdal
Nordic Bioscience A/S, Herlev, Denmark. jsa@nordicbioscience.com.
Jannie Mb
JM
Sand
Nordic Bioscience A/S, Herlev, Denmark. nwi@nordicbioscience.com.
Nicholas
N
Willumsen
Boehringer-Ingelheim Pharma GmbH, Biberach, Germany. claudia.diefenbach@boehringer-ingelheim.com.
Claudia
C
Diefenbach
Department of Systems Biology, The Technical University of Denmark, Kgs. Lyngby, Denmark. bis@bio.dtu.dk.
Birte
B
Svensson
Department of Systems Biology, The Technical University of Denmark, Kgs. Lyngby, Denmark. ph@bio.dtu.dk.
Per
P
Hägglund
Nordic Bioscience A/S, Herlev, Denmark. djl@nordicbioscience.com.
Diana J
DJ
Oersnes-Leeming
D000230:Adenocarcinoma / Q000378:metabolism*; D000368:Aged; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D016022:Case-Control Studies; D004549:Elastin / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis; D019272:Leukocyte Elastase / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D008175:Lung Neoplasms / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D010446:Peptide Fragments / Q000378:metabolism*; D055752:Small Cell Lung Carcinoma / Q000378:metabolism*
D010446:Peptide Fragments; D004549:Elastin; D019272:Leukocyte Elastase