BOTULINUM NEUROTOXIN
Structural diversity of botulinum neurotoxins
Raymond C. Stevens, The Scripps Research Institute
Abstract:
The botulinum neurotoxins, BoNTs, produced by Clostridium botulinum are among the most potent toxins known to man. They are classified as potential bioweapon, Category A, because of their extreme potency and lethality, their ease of production and transport, and the need for prolonged intensive care among affected persons. The overall goal of this project is to characterize the various serotypes and subtypes of BoNTs to characterize these variants at the DNA and protein level and develop a deeper understanding of neurotoxin action. We will focus on three major research areas: (1) continuing to deepen our understanding of the cell receptor recognition and binding processes as well as the translocation process by determining the structures of the toxins in complex with cell recognition receptor molecules; (2) developing a detailed understanding on how the recently developed antibodies (Abs), work and how they can be improved; and (3) using structure-based approaches to engineer the next generation broad spectrum diagnostic antibodies (working towards one antibody to recognize multiple toxin subtypes and serotypes). The study will characterize a number of BoNT variants to assess their impact on the development of diagnostics and therapeutics and to understand differences in biological action. In addition to the structures, the project will also produce highly purified protein samples that will be made available to collaborators and other workers in the biodefense consortium.
Evolving diagnostic antibodies for botulinum neurotoxins
James Marks, UC San Francisco
Abstract:
The botulinum neurotoxins, BoNTs, produced by Clostridium botulinum are among the most potent toxins known to man. They are classified as potential bioweapon, Category A, because of their extreme potency and lethality, their ease of production and transport, and the need for prolonged intensive care among affected persons. This project focuses on generating monoclonal antibodies to be used for the development of diagnostic assays and devices as well as general tools for understanding toxin structure-function relationships. The goal is to generate mAbs that recognize all neurotoxin subtypes multiple serotypes. Studies will be conducted to understand the role of antibody epitopes on toxin recognition and to use this knowledge to engineer mAbs allowing more sensitive toxin detection or greater therapeutic efficacy. Antibodies will be generated against the heavy chain domains to better understand the receptor recognition and binding process and against the light chain domain for use in assays and diagnostic devices. Samples will be made available to collaborators as well as to others in the biodefense consortium.
Fluorescent and bioluminescent assays for botulinum neurotoxins
Markus Kalkum, City of Hope Medical Center
Abstract:
This project will develop novel biochemical methods and devices for the attomolar detection of all 7 serotypes and subtypes of botulinum neurotoxin (BoNT), except type G, using assays with a large immuno-sorbent surface area (ALISSA). Based on our preliminary data, we hypothesize that it is feasible to generate both fluorescent and bioluminescent substrates that are resistant to non-BoNT proteolytic cleavage, but can rapidly be activated by BoNTs. The specific aims of the proposed research are to: 1) develop novel fluorogenic substrates for BoNT serotypes A to F by creating peptide libraries with proteinogenic and non-proteinogenic amino acids that resist non-BoNT proteases and are efficiently cleavaged by BoNTs in serum, solid organ extracts, enema, stool and food; 2) generate recombinant luciferase-based luminogenic protein substrates that become activated by reactions mediated by BoNTs; and 3) apply existing and novel BoNT ALISSAs to measure toxin distribution in animal sera and organs. A multidisciplinary and multi-institutional research team will work together to: 1) study sublethally BoNT-intoxicated mice to determine pharmacokinetics and serotype-dependent toxin distributions in organs following parenteral or oral intoxication; 2) develop and test fully automated benchtop devices for the quantitative detection of BoNTs at attomolar sensitivity; and 3) test and validate the assays with complex biological matrices such as serum, solid organ and food extracts and clinical samples.
Benchtop device for the detection of botulinum neurotoxins in clinical samples
Dobrin Nedelkov, Intrinsic Bioprobes, Inc.
Abstract:
We will develop devices for the quantitative detection of botulinum neurotoxins (BoNT) at attomolar sensitivity with fully automated bench-top instrumentation. We will adopt the existing bead-based BoNT assays with a large immuno-sorbent surface area (ALISSA) technology into a monolithic microcolumn format to be utilized by common liquid handling robots. We will also develop an automated, highly sensitive, and inexpensive wave-guide-based optical read-out system for integration in the robots. The resulting functional prototype system is expected to automatically process and analyze multiple samples simultaneously and repeatedly. Our assay system will be tested and validated with complex biological matrices such as serum, solid organ and food extracts and clinical samples.
