An innovative high sensitivity tool for active neutrophil elastase quantification

Abstract

<b>Introduction:</b> The serine protease, neutrophil elastase (NE), is widely recognised as a key biomarker in respiratory disease; with its potential therapeutic value illustrated by the volume of anti-NE therapies currently undergoing clinical evaluation. However, recruitment for these studies is constrained as many patients are ‘non-producers’ of sputum. Alternative sample types include nasal fluid but are reputed to contain lower NE levels. To address this issue, ProAxsis Ltd has harnessed its existing technology to produce an activity-based immunoassay (ABI) for active NE quantification, with enhanced sensitivity. <b>Aims and Objectives:</b> To amplify the existing sensitivity of ProAxsis’ ProteaseTag® Active NE Immunoassay by refinement of key binding reagents and through transfer to a luminescence-based detection system. <b>Methods:</b> NE ProteaseTag®, PRX1138, was synthesised using a combination of solid-phase peptide synthesis (SPPS), and solution-phase methodologies, with a monoclonal anti-NE antibody generated by hybridoma technology. Binding reagents were then incorporated into an ABI format with a chemiluminescent substrate used for the detection step. <b>Results:</b> A 20-fold increase in sensitivity, compared to the original NE ABI, was achieved following completion of these modifications; with a lower limit of quantification in the picogram range observed. Further enhancement in sensitivity may be obtained once optimisation of other reagents is performed. <b>Conclusions:</b> The development of a high sensitivity ABI for NE raises the possibility of improving patient recruitment for clinical trials by enabling the analysis of surrogate sample types, such as nasal fluid.