A bacteriophage-based platform for rapid trace detection of proteases.
Sensitive, inexpensive, and rapid protease activity assays are of great merit for clinical diagnostics. Detection of protease-based toxins produced by Clostridium botulinum and Bacillus anthracis represents a particularly challenging task, as exceptional sensitivity is a prerequisite because of the extreme potency of the toxins. Here we present an inexpensive and sensitive assay platform for activity-based protease quantification utilizing filamentous bacteriophage as an exponentially amplifiable reporter and its application to the detection of these bacterial toxins. The assay is based on specific cleavage of bacteriophage from a solid support and its subsequent quantification by means of infectivity or quantitative PCR. Detection of botulinum neurotoxin (BoNT) serotypes A and B and anthrax lethal factor in the picomolar range was demonstrated with a limit of detection of 2 pM for BoNT/A under optimized conditions.
Capek
Petr
P
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Kirkconnell
Killeen S
KS
Dickerson
Tobin J
TJ
D001435:Bacteriophages / Q000737:chemistry*; D057230:Limit of Detection; D010447:Peptide Hydrolases / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010447:Peptide Hydrolases
Physiol Rev. 2000 Apr;80(2):717-66
10747206
Anal Biochem. 2006 Apr 1;351(1):84-92
16500606
ACS Nano. 2009 Nov 24;3(11):3567-80
19860382
Biochimie. 2010 Nov;92(11):1689-704
20470858
Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):3978-82
16537471
Methods Mol Biol. 1996;58:335-9
8713881
Mol Aspects Med. 2009 Dec;30(6):431-8
19665472
Toxins (Basel). 2010 Jan;2(1):24-53
22069545
Biol Chem. 2002 Jul-Aug;383(7-8):1107-12
12437093
Nat Biotechnol. 2003 Jan;21(1):86-9
12469133
Adv Clin Chem. 2005;40:219-59
16355924