NE ULTRA – A NOVEL PROTEASETAG® FOR THE INHIBITION AND DETECTION OF NEUTROPHIL ELASTASE IN BIOLOGICAL SAMPLES

Introduction: Neutrophil elastase (NE) is a serine protease released from the azurophilic granules in leukocytes (or neutrophils). Its primary role is to assist in the proteolytic breakdown and destruction of phagocy-tosed foreign pathogens as part of the normal immune response. However, secreted active NE has been implicated in contributing to a range of inflam-matory disorders including rheumatoid arthritis, chronic obstructive pul-monary disease (COPD) and cystic fibrosis (CF). ProAxsis has developed a range of ProteaseTags® which are able to rapidly and selectively bind to active proteases. The incorporation of such ProteaseTags® within activi-ty-dependent immunoassays enables the quantification of active proteases within biological samples and has resulted in the recent commercialisation of our ProteaseTag® Active Neutrophil Elastase Immunoassay.Aims: Here we report the application and benefits of NE Ultra, a novel biotinylated ProteaseTag® targeting NE. Furthermore, we compare it with another commercially available irreversible NE inhibitor MeO-Suc-AAPV-CH2Cl in terms of selectivity and stability.Methods: Kinetic evaluation and selectivity profiling of NE Ultra and MeOSuc-AAPV-CH2Cl against NE and other proteases, including thrombin, cathepsin G, cathepsin B and chymotrypsin was determined by fluorogenic steady-state enzyme assays. The stability of NE Ultra and MeOSuc-AAPV-CH2Cl was compared by overnight incubation in 10% (v/v) serum from CF patients, followed by an NE activity assay. Further-more, the application of NE Ultra as an activity-based probe (ABP) for the detection of NE in CF sol samples was carried out through SDS-PAGE and electroblotting techniques.Results: NE Ultra and MeOSuc-AAPV-CH2Cl showed excellent irre-versible inhibition of NE with similar potency displaying k3/Ki values of 4.08 (± 1.84) x 106 M-1 min-1 and 6.68 (± 1.62) x 106 M-1 min-1, respectively. NE Ultra displayed excellent selectivity for NE, having no effect on the activity of other proteases including cathepsin G, thrombin and cathepsin B. In contrast, MeOSuc-AAPV-CH2Cl completely abrogated the activity of cathepsin B. In terms of stability, following overnight incubation both NE Ultra and MeOSuc-AAPV-CH2Cl displayed inhibitory action against NE, however, MeOSuc-AAPV-CH2Cl showed a slight reduction in potency suggesting a lower stability in biological samples. When NE Ultra was examined as an ABP, it enabled streptavidin-mediated detection of NE in CF sol samples, following separation and detection via SDS-PAGE and electroblotting.Conclusion: We report the application of a novel ProteaseTag®, NE Ultra, as an irreversible inhibitor and potent ABP for NE. This affinity label has a number of advantages over other commercially available irreversible NE inhibitors such as MeOSuc-AAPV-CH2Cl, in terms of improvements in stability and selectivity as well as the ability to visualise active NE in clinical samples, when used in combination with a streptavidin-conjugate. NE Ultra has therefore the potential for broad application across laborato-ry-based CF research.