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01.12.2015 | Research article | Ausgabe 1/2015 Open Access

BMC Pulmonary Medicine 1/2015

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

Zeitschrift:
BMC Pulmonary Medicine > Ausgabe 1/2015
Autoren:
Jacob H Kristensen, Morten A Karsdal, Jannie MB Sand, Nicholas Willumsen, Claudia Diefenbach, Birte Svensson, Per Hägglund, Diana J Oersnes-Leeming
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12890-015-0048-5) contains supplementary material, which is available to authorized users.

Competing interests

JHK, MAK and DJOL have filed the patent 1400472.5 'Elastin lung disease diagnostics'. The remaining authors declare that they have no competing interests.

Authors’ contributions

JHK contributed to the design of the immunoassay and performed all laboratory procedures, calculated the results, performed the statistical analysis and drafted the manuscript; MK contributed to the design of the immunoassay, data interpretation, manuscript draft and manuscript revision; JMBS contributed to statistical analysis, data interpretation and manuscript revision; NWI contributed to data interpretation and manuscript revision; CD contributed to data analysis and manuscript revision; BS contributed to manuscript revision, PH contributed to manuscript revision; DJL contributed to the design of the immunoassay; data interpretation, manuscript draft and manuscript revision. All authors read and approved the final manuscript.

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.
Zusatzmaterial
Additional file 1: Appendix 1, Table S1: Patient Demographics and Clinical Profiles of Cancer and IPF Patients.
12890_2015_48_MOESM1_ESM.pdf
Additional file 2: Appendices 1-2, Figure S1 and Figure S2. Appendix 1: EL-NE-B Methods; Appendix 2: EL-NE-B Data; Figure S1: Characterization and specificity of the EL-NE-B monoclonal antibody; Figure S2: The EL-NE-B Assay in IPF.
12890_2015_48_MOESM2_ESM.pdf
Literatur
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