Attomolar detection of botulinum toxin type A in complex biological matrices.
BACKGROUND
A highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT) in complex biological samples such as foods or serum is desired in order to 1) counter the potential bioterrorist threat 2) enhance food safety 3) enable future pharmacokinetic studies in medical applications that utilize BoNTs.


RESULTS
Here we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the "gold standard" mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity.


CONCLUSIONS
The sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications.
Bagramyan
Karine
K
Immunology Division, Beckman Research Institute of the City of Hope, Duarte, California, United States of America.
Barash
Jason R
JR
Arnon
Stephen S
SS
Kalkum
Markus
M
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D019274:Botulinum Toxins, Type A / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D051379:Mice
D019274:Botulinum Toxins, Type A