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Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis
Author(s) -
Lefkowitz Roy B.,
SchmidSchönbein Geert W.,
Heller Michael J.
Publication year - 2010
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201000011
Subject(s) - trypsin , chromatography , electrophoresis , chemistry , whole blood , fluorescence , substrate (aquarium) , enzyme , biochemistry , enzyme assay , gel electrophoresis , cleavage (geology) , polyacrylamide gel electrophoresis , microbiology and biotechnology , biology , ecology , paleontology , physics , quantum mechanics , fracture (geology) , immunology
The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge‐changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly‐ L ‐glutamic acid‐doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 μL (0.3 ng/mL) in whole human blood was achieved after a 1‐h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50‐ to 200‐fold better than the estimated reference levels for trypsin (15–60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point‐of‐care diagnostics.