Botulinum toxin is an extraordinarily potent molecule that has an unusually long duration of action. Despite this, there is little information available on natural mechanisms for metabolism or elimination and virtually no information on pharmacologically induced mechanisms for metabolism and elimination. Therefore, a number of experiments were performed on laboratory animals that addressed two major issues: 1) the effect of blood on the structure, function, and biologic half-life of the toxin, and 2) the effect of neutralizing antibodies on half-life and elimination of circulating toxin. In the first series of studies, the metabolic transformation of toxin was assessed by incubating it in blood for varying lengths of time. At each time point, aliquots were examined to determine the amount of toxin, the structure of toxin, the catalytic activity of toxin, and the neuromuscular blocking activity of toxin. This work demonstrated that blood did not alter any characteristic of the toxin molecule. Experiments were also done in which toxin was administered to mice and rats at doses that produced clinical poisoning. The results demonstrated that the elimination half-life for native (nonmetabolized) toxin in blood and serum was 230 to 260 min. During the second series of studies, the rate of elimination of circulating toxin was studied in the presence of antibodies directed against the carboxyl-terminal half of the toxin molecule. This work demonstrated that neutralizing antibodies 1) enhanced clearance of toxin from the circulation and 2) enhanced tissue accumulation of toxin, particularly in liver and spleen.
Most reports dealing with vaccines against botulinum toxin have focused on the injection route of administration. This is unfortunate, because a mucosal vaccine is likely to be more efficacious for patients and pose fewer risks to health care workers and to the environment. Therefore, efforts were made to generate a mucosal vaccine that provides protection against the botulinum serotypes that typically cause human illness (serotypes A, B, and E). This work demonstrated that carboxy-terminal peptides derived from each of the three serotypes were able to bind to and penetrate human epithelial barriers in vitro, and there was no cross inhibition of membrane binding and transcytosis. The three polypeptides were then tested in vivo as a trivalent vaccine that could be administered to mice by the intranasal route. The results indicated that the mucosal vaccine evoked high secretory titers of immunoglobulin A (IgA), as well as high circulating titers of IgG and IgA, and it also evoked a high level of resistance to challenge with toxin. The immunoglobulin responses and the levels of resistance to challenge were increased by coadministration of adjuvants, such as chitosan and vitamin E. At least three mechanisms were identified to account for the antibody-induced resistance: (i) blockade of toxin absorption across epithelial cells, (ii) enhanced clearance of toxin from the circulation, and (iii) blockade of toxin action at the neuromuscular junction. These results are a compelling demonstration that a mucosal vaccine against multiple serotypes of botulinum toxin has been identified.
We present an innovative centrifugal
microfluidic immunoassay platform
(SpinDx) to address the urgent biodefense and public health need for
ultrasensitive point-of-care/incident detection of botulinum toxin.
The simple, sample-to-answer centrifugal microfluidic immunoassay
approach is based on binding of toxins to antibody-laden capture particles
followed by sedimentation of the particles through a density-media
in a microfluidic disk and quantification by laser-induced fluorescence.
A blind, head-to-head comparison study of SpinDx versus the gold-standard
mouse bioassay demonstrates 100-fold improvement in sensitivity (limit
of detection = 0.09 pg/mL), while achieving total sample-to-answer
time of <30 min with 2-μL required volume of the unprocessed
sample. We further demonstrate quantification of botulinum toxin in
both exogeneous (human blood and serum spiked with toxins) and endogeneous
(serum from mice intoxicated via oral, intranasal, and intravenous
routes) samples. SpinDx can analyze, without any sample preparation,
multiple sample types including whole blood, serum, and food. It is
readily expandable to additional analytes as the assay reagents (i.e.,
the capture beads and detection antibodies) are disconnected from
the disk architecture and the reader, facilitating rapid development
of new assays. SpinDx can also serve as a general-purpose immunoassay
platform applicable to diagnosis of other conditions and diseases.
The ability of botulinum toxin to poison cholinergic nerve transmission is a dynamic phenomenon that involves not only the actions of the toxin on the body but also the actions of the body on the toxin. The former has been the subject of intense research, whereas the latter has received almost no attention.
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