The development of a novel immunoassay for the quantification of active tryptase

Abstract

Introduction: Tryptase, a serine protease found in the secretory granules of mast cells, has been associated with several diseases including allergic asthma and Chronic Obstructive Pulmonary Disease (COPD). Elevated levels of tryptase occur in biological fluids, including blood, following mast cell degranulation, in response to allergic stimulation. In COPD, tryptase levels in sputum correlate with the severity of disease. Aims and Objectives ProAxsis has developed a range of ProteaseTags ® which selectively bind to the active forms of proteases, only. When combined with an appropriate antibody, they provide activity-based immunoassays (ABIs) for the quantification of active proteases. Here we report the development of a novel ProteaseTag ® ABI for the quantification of active mast cell tryptase. Methods: A ProteaseTag ® , designed to irreversibly inhibit mast cell tryptase, was synthesised using methodologies developed in-house. The desired irreversible inhibition of tryptase was established kinetically using a fluorogenic activity assay and confirmed with electroblotting techniques. The ProteaseTag ® was subsequently incorporated into an ABI, which was evaluated for the quantification of active tryptase. Results: Our novel tryptase ProteaseTag ® ABI proved successful for the quantification of active tryptase, with detection limits ranging from 7.8125 ng/ml to 500 ng/ml. Conclusions We have developed a novel ABI for the measurement of active tryptase, which could allow rapid quantification of the protease in patient samples. The use of our ABI could facilitate the adoption of active tryptase as a validated biomarker in several respiratory diseases, including asthma and COPD.