Utilisation of a novel ProteaseTag™ activity immunoassay for the specific measurement of neutrophil elastase in patients with Cystic Fibrosis

Kelly Moffitt, Brian Walker, Joseph Elborn, Lorraine Martin

Research output: Contribution to conferencePoster

Abstract

Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including cystic fibrosis (CF) however, its detection and quantification in biological samples is confounded by a lack of robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex samples containing multiple proteolytic and hydrolytic enzymes, resulting in an over-estimation of the target protease. ELISA systems measure total protein levels which can be a mixture of latent, active and protease-inhibitor complexes. We have therefore developed a novel immunoassay (NE-Tag ELISA), incorporating an activity dependent ProteaseTag™ and a specific antibody step, which is selective and specific for the capture of active NE. The objective of this study was to clinically validate NE-Tag ELISA for the detection of active NE in sputum from CF patients. Sputum (n=45) was recovered from CF patients hospitalised for acute exacerbation. Sol was recovered and analysed for NE activity using the NE-Tag ELISA and two fluorogenic substrate-based assays [1. Suc-AAPV-AMC (Sigma) and 2. InnozymeTM Immunocapture assay (Calbiochem)]. NE activity between assays and with a range of clinical parameters was correlated.A highly significant correlation was shown between assays. NE activity (NE-Tag) further correlated appropriately with clinical parameters: inversely with FEV1 (p = 0.036) and positively with CRP (p = 0.035), neutrophils and total white cell counts (p < 0.001). The InnozymeTM assay showed similar correlations with the clinical parameters (with the exception of CRP). No correlations with any of the clinical parameters were observed when NE was measured using the standard fluorogenic substrate.
Original languageEnglish
Publication statusPublished - 14 Apr 2015
EventProteinase 2015: 9th RSC / SCI symposium on Proteinase Inhibitor Design - Novartis Campus, Basel, Switzerland
Duration: 13 Apr 201514 Apr 2015

Conference

ConferenceProteinase 2015: 9th RSC / SCI symposium on Proteinase Inhibitor Design
CountrySwitzerland
CityBasel
Period13/04/201514/04/2015

Keywords

  • neutrophil elastase
  • cystic fibrosis
  • inflammation
  • proteases
  • biomarker
  • Immunoassay
  • ELISA

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