Whole blood assay for elastase, chymotrypsin, matrix metalloproteinase-2, and matrix metalloproteinase-9 activity.
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.
Lefkowitz
Roy B
RB
Department of Bioengineering, University of California San Diego, La Jolla, California 92093-0448, USA.
Schmid-Schönbein
Geert W
GW
Heller
Michael J
MJ
D002918:Chymotrypsin / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000097:blood*; D020780:Matrix Metalloproteinase 9 / Q000097:blood*; D010196:Pancreatic Elastase / Q000097:blood*; D010455:Peptides / Q000737:chemistry; D019095:Point-of-Care Systems; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
D010455:Peptides; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9