Whole blood assay for elastase, chymotrypsin, matrix metalloproteinase-2, and matrix metalloproteinase-9 activity

Roy B. Lefkowitz, Geert W. Schmid-Schönbein, Michael (Mike) Heller

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

The ability to measure protease activity in the blood is important for the development of future diagnostics and for biomedical research. Presently, protease assays require sample preparation, making them time-consuming, costly, less accurate, and unsuitable for point-of-care (POC) diagnostics. Recently, we demonstrated a unique method for measuring clinically relevant levels of trypsin activity in only a few microliters of whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target protease. Using a simple electrophoretic format, the fragments could be rapidly separated, concentrated, and detected directly from a whole blood sample. We now report on the development of new protease substrates for the measurement of elastase, chymotrypsin, matrix metalloproteinase (MMP)-2, and MMP-9 activity in whole blood. In these studies, detection limits ranging from 1 to 40 pg in 6 μL of 1- phosphate-buffered saline (PBS) (0.2′6 ng/mL) were achieved after a only 1 h reaction of enzyme and substrate. In subsequent experiments measuring spiked protease in whole blood (with endogenous protease present), detection limits ranging from 100 to 200 ng/mL were achieved after a 1 h reaction. Thus, these new substrates demonstrate broad applicability toward clinically relevant detection of important disease-relevant proteases.

Original languageEnglish (US)
Pages (from-to)8251-8258
Number of pages8
JournalAnalytical Chemistry
Volume82
Issue number19
DOIs
StatePublished - Oct 1 2010
Externally publishedYes

Fingerprint

Pancreatic Elastase
Matrix Metalloproteinase 2
Matrix Metalloproteinase 9
Chymotrypsin
Assays
Blood
Peptide Hydrolases
Substrates
Trypsin
Phosphates
Peptides
Enzymes

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Whole blood assay for elastase, chymotrypsin, matrix metalloproteinase-2, and matrix metalloproteinase-9 activity. / Lefkowitz, Roy B.; Schmid-Schönbein, Geert W.; Heller, Michael (Mike).

In: Analytical Chemistry, Vol. 82, No. 19, 01.10.2010, p. 8251-8258.

Research output: Contribution to journalArticle

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