Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis

Roy B. Lefkowitz, Geert W. Schmid-Schönbein, Michael J. Heller

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2442-2451
Number of pages10
JournalELECTROPHORESIS
Volume31
Issue number14
DOIs
StatePublished - Jul 1 2010

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Keywords

  • Charge-changing substrate
  • Detection
  • Polyanionic focusing gel
  • Trypsin
  • Whole blood assay

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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