|Abstract Title:||Detection and differentiation of botulinum neurotoxins for the diagnosis and prevention of botulism|
|Presenter Name:||Dr John Barr|
|Co-authors:||Dr Suzanne Kalb|
|Company/Organisation:||Centers for Disease Control and Prevention (CDC)|
|Session Choice:||Clinical Hyphenations|
Abstract Information :
Botulinum neurotoxins (BoNTs) are the most poisonous substances known to mankind and cause the disease botulism. The analysis of trace amounts of protein toxins in highly complex matrices is an analytical challenge. We have developed and implemented a sensitive method that uses the enzymatic amplification of products with mass spectrometry to allow detection of BoNT and determine its serotype (A-G) in low attomole/mL. Furthermore, we have performed detailed proteomics analysis to aid epidemiologic investigations discovering sources of intoxication and commonality/differences between concurrent botulism outbreaks.
This method has three levels of selectivity including immunomagnetic extraction with monoclonal antibodies, enzymatic activity of the toxin on a peptide substrate that mimics the toxins natural, in vivo target, and specific detection of product peptides with MALDI TOF MS. The subtype and toxin variant are then identified by digesting the bead mixture with trypsin/chymotrypsin followed by nLC-MS/MS on a high resolution hybrid mass spectrometer.
The mass spectrometry-based BoNT analysis can detect and differentiate all seven serotypes. The detection limits are less than 0.1 mouse LD50 (low attomole/mL) in stool, serum, and food and this method is much more rapid than the traditional mouse bioassay. The method has been used to investigate many botulism outbreaks and has confirmed botulism for foodborne, infant, adult colonization, and wound botulism. The addition of subtyping the toxins has added a new layer of information that has been used in several outbreaks across the United States.
The new BoNT method has allowed for more sensitive and rapid conformation of botulism. Serum now is routinely tested and we are now able to routinely detect BoNT in wound botulism cases. The subtyping has provided rapid information for botulism investigations and has provided a deeper understanding to the diversity of subtypes that are causing human botulism in the United States.