Introduction and ObjectivesSputum levels of active neutrophil elastase (NE) are frequently elevated in respiratory diseases, such as bronchiectasis, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF). Increased NE levels have been demonstrated to inversely correlate with pulmonary function, and risk of exacerbations. Active NE is also recognised as a biomarker of subclinical infection; however, no tools presently exist for its routine monitoring at home or in the clinic. Translation of the ProAxsis NE Activity Based Immunoassay (ABI) to a Point of Care (PoC) device, would facilitate routine monitoring of those patients at highest risk of upcoming exacerbations; enabling pre-emptive medical intervention, and mitigating the patient’s risk of developing serious complications.MethodsActive NE levels in sputum samples (n=10) were determined using the NE ABI (ProAxsis Ltd, Belfast), in accordance with manufacturer’s instructions; followed by assessment using the NEATstik PoC test (threshold 10 µg/ml in sputum). For measurement of NE using NEATstik, sputum was diluted x10, gently rotated for 1 min, an aliquot (70 µl) was then transferred onto the test sample port of the device and allowed to develop for 10 min, after which the signal intensity at the test-line was visually graded (0–10).ResultsNE ABI analysis of sputum revealed that 7 out of 10 samples under investigation contained active NE at levels above the NEATstik test threshold. All 7 samples were found to produce a strong positive test line on the PoC device. Moreover, no test line was visible for the remaining samples with active NE concentrations below 10 µg/ml.ConclusionAvailability of NEATstik, the first highly sensitive and specific PoC test for the rapid detection of active NE in complex clinical samples, should enable the proactive management of multiple chronic respiratory diseases. It has the potential to assist in the identification of patients at highest risk of imminent exacerbations, and thus allow closer monitoring by their clinical team and/or pre-emptive treatment to avoid/minimise the impact of such exacerbations. Additionally, for those presenting with an ongoing exacerbation, the test facilitates patient stratification, with those most likely to respond to antibiotic therapy identified and their response to treatment assessed.
Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including chronic obstructive pulmonary disease (COPD). However, it’s detection and quantification in biological samples is confounded by a lack of reliable and robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex clinical samples containing multiple proteolytic and hydrolytic enzymes which have the ability to hydrolyse the substrate, thereby resulting in an over-estimation of the target protease. Furthermore, ELISA systems measure total protease levels which can be a mixture of latent, active and protease-inhibitor complexes. Therefore, we have developed a novel immunoassay (ProteaseTag™ Active NE Immunoassay) which is selective and specific for the capture of active NE in sputum and Bronchoalveolar Lavage (BAL) in patients with COPD.The objective of this study was to clinically validate ProteaseTag™ Active NE Ultra Immunoassay for the detection of NE in sputum from COPD patients.20 matched sputum sol samples were collected from 10 COPD patients (M = 6, F = 4; 73 ± 6 years) during stable and exacerbation phases. Samples were assayed for NE activity utilising both ProteaseTag™ Active NE Ultra Immunoassay and a fluorogenic substrate-based kinetic activity assay.Both assays detected elevated levels of NE in the majority of patients (n = 7) during an exacerbation (mean = 217.2 µg/ml ±296.6) compared to their stable phase (mean = 92.37 µg/ml ±259.8). However, statistical analysis did not show this difference to be significant (p = 0.07, ProteaseTag™ Active NE Ultra Immunoassay; p = 0.06 kinetic assay), which is highly likely to be due to the low study number. A highly significant correlation was found between the 2 assay types (p ≤ 0.0001, r = 0.996).NE as a primary efficacy endpoint in clinical trials or as a marker of inflammation within the clinic has been hampered by the lack of a robust and simple to use assay. ProteaseTag™ Active NE Immunoassay specifically measures only active NE in clinical samples, is quick and easy to use (<3 h) and has no dependency on a kinetic readout. ProteaseTag™ technology is currently being transferred to a lateral flow device for use at Point of Care.
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