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United States Department of Agriculture

Agricultural Research Service

Research Project: Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins

Location: Foodborne Toxin Detection and Prevention

Title: Substrates and controls for the quantitative detection of active botulinum neurotoxin in protease-containing samples

Authors
item Bagramyan, Karine
item Kaplan, Bruce
item Cheng, Luisa Wai Wai
item Rummel, Andreas
item Kalkum, Marcus

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 5, 2013
Publication Date: June 4, 2013
Citation: Bagramyan, K., Kaplan, B.E., Cheng, L.W., Rummel, A., Kalkum, M. 2013. Substrates and controls for the quantitative detection of active botulinum neurotoxin in protease-containing samples. Analytical Chemistry. 85(11):5569-76. doi: 10.1021/ac4008418.

Interpretive Summary: The study of the activity of botulinum neurotoxins(BoNT)inside animals requires the ability to detect minuscule amounts of toxin in blood or other body fluids and is therefore very challenging because very little toxin can cause death quickly. In this study, the new sensitive technique called “Assay with a Large Immunosorbent Surface Area” (ALISSA)is used to detect active BoNT found inside animals. New reagents were developed to diminish the background signals that previously prevented detection of BoNT in body fluids or wastes. We were able to detect attomolar amounts of BoNT in animal blood over time, and determined the mouse clearance rate of BoNT serotype A after oral feeding.

Technical Abstract: Botulinum neurotoxins (BoNTs) enjoy a wide variety of medical applications. However, limited pharmacokinetic data on active BoNT is available. Monitoring BoNT activity in the circulation is a challenging task, due to BoNT’s enormous toxicity, rapid neuronal uptake, and removal from the bloodstream. Previously we reported a sensitive BoNT “Assay with a Large Immunosorbent Surface Area” (ALISSA) that measures the intrinsic endopeptidase activity of bead-captured BoNT by conversion of fluorogenic peptide substrates. However, in complex biological samples, protease contaminants can also cleave the substrates, reducing sensitivity and specificity of the assay. Here, we present a novel set of fluorogenic peptides with either green or blue light emitters that serve as BoNT-specific substrates and protease-sensitive controls. BoNT-cleavable substrates contain a C-terminal norleucine, and BoNT-non-cleavable controls, 6-aminohexanoic acid. The substrates are cleaved by all BoNT/A subtypes with the exception of A4. Substrates and control peptides can be cleaved by other non-BoNT proteases, such as trypsin, proteinase K, and thermolysin, while obeying Michaelis-Menten kinetics. Using our novel substrate/control set, we studied BoNT/A1 activity in intravenous and oral mouse intoxication models. Intravenous injection of BoNT/A1 complex in decreasing amounts from 100 to 4pg/mouse led to detectable serum activities ranging from ~3600 to 10 attomol/L after 1h. Endopeptidase activity of 1 µg orally administered BoNT complex became detectable in blood after 5 h and reached a maximum at 7 h. Active toxin redistribution, t½a, was 46 ± 12 min, and clearance t½ß was 458 ± 57 min, confirming the utility of the approach for pharmacokinetic studies.

Last Modified: 10/21/2014
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