Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling

BMC Pulm Med. 2015 May 3;15:53. doi: 10.1186/s12890-015-0048-5.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / metabolism*
  • Aged
  • Carcinoma, Squamous Cell / metabolism*
  • Case-Control Studies
  • Elastin / metabolism*
  • Enzyme-Linked Immunosorbent Assay
  • Extracellular Matrix / metabolism*
  • Female
  • Humans
  • Idiopathic Pulmonary Fibrosis
  • Leukocyte Elastase / metabolism*
  • Lung / metabolism*
  • Lung Neoplasms / metabolism*
  • Male
  • Middle Aged
  • Peptide Fragments / metabolism*
  • Small Cell Lung Carcinoma / metabolism*

Substances

  • Peptide Fragments
  • Elastin
  • Leukocyte Elastase