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A3676 - Augmentation of ProteaseTag® Active Neutrophil Elastase Immunoassay Sensitivity
Author Block: D. McCafferty, K. L. Moffitt, C. Robb, A. Kennedy, D. Ramsay, T. Ferguson, B. Walker; ProAxsis Ltd, Belfast, United Kingdom.
Rationale: Sputum levels of active neutrophil elastase (NE) are frequently elevated in respiratory diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and bronchiectasis; and have been demonstrated to correlate inversely with pulmonary function, and risk of future exacerbations. Consequently, the development of anti-elastase therapies continues to pique the interest of the pharmaceutical industry; with numerous compounds undergoing clinical trials at present. However, recruitment for these studies is severely constrained, since many patients, from both healthy and disease populations, are ‘non-producers’ of sputum. Alternative sample types include exhaled breath condensate (EBC) and nasal fluid, but are estimated to contain lower levels of active NE. To address this issue, ProAxsis Ltd has harnessed its existing ProteaseTag® technology to produce an activity-based immunoassay (ABI), for NE quantification, with enhanced sensitivity. Such a tool could facilitate the assessment of novel anti-elastase therapeutics, based on data collected from a larger cohort of study subjects. Methodology: NE ProteaseTag® was synthesised using a combination of solid-phase peptide synthesis (SPPS), and solution-phase methodologies. Inhibitory potency, against active human NE, was examined using the fluorogenic peptide substrate MeoSuc-Ala-Ala-Pro-Val-AMC (Merck); followed by assessment of the interaction between ProteaseTag® and NE (reversible/irreversible) by Western immunoblotting. NE ProteaseTag® was subsequently incorporated into an ABI format for the specific measurement of lower concentrations of active NE. Results: NE ProteaseTag® exhibited potent and rapid irreversible inactivation of active human NE in fluorescent activity assays, with a high second order rate constant determined (4.24 (± 2.35) x 106 M-1 Min-1); a pattern confirmed following Western immunoblotting. Furthermore, upon incorporation of NE ProteaseTag® into an ABI format, active NE was readily measurable in under 3 hours, with detection limits ranging from 0.975 ng/ml to 62.5 ng/ml; equating to a 16-fold increase in sensitivity, relative to ProAxsis’ original ProteaseTag® NE ABI. Conclusions: Active NE, within sputum, is frequently employed as the primary endpoint in clinical trials, designed to evaluate the effectiveness of anti-elastase therapies. However, it is anticipated that these research studies could be further improved, through the inclusion of surrogate sample types, such as EBC and nasal fluid. Here we report the development of a more sensitive ProteaseTag® ABI for NE detection and quantification; which may have utility for analysis of clinical samples, suspected to contain lower concentrations of active NE.