Botulinum Assay

The detection of flu viruses via a fluorescent sandwich immunoassay was reported by Bucher.(10) However, the method sensitivity was too low for direct detection of the virus. A novel sandwich immunoassay was described by Ogcr((lff7 for the detection of Botulinum Toxin A. Antibodies specific for Clostridium botulinum were covalently attached to the surface of a tapered fiber. After the capture of the antigen, a sandwich was formed with a rhodamine-labeled anti-toxin IgG, and the evanescent wave was measured. The assay was highly specific with detection limits near 5 ppb. 

Molecular weight of the main bacterial toxins ranges from 28,000 to 150,000, which makes it possible for most sensitive SPR biosensors to measure their concentrations directly or using a sandwich assay. Examples of food safety-related toxins detected by SPR biosensors include Botulinum toxin (detection limit 2.5 pg/ml " ), . coli enterotoxin (detection limit 6 pg/ml " ) and Staphylococcal enterotoxin B (detection limit 5 ng/ml and 0.5 ng/ml for direct detection and sandwich assay, respectively" ). 

Fach, P., Perelle, S., Dilasser, F., Grout, J., Dargaignaratz, C., Botella, L., Gourreau, J.-M., Carlin, F., Popoff, M.R. and Broussolle, V., Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in Northern France, Appl. Em. Microbiol., 68, 5870-5876, 2002. 

Clostridium botulinum neurotoxin, the most effective toxin known to date, with a mice lethal dose of about 50 pg/mL (330 fmol/mL) was the target antigen in IPCR assays developed by Wu et al. [48] and Chao et al. [88]. In these assays, detection limits of 5 fg (33 amol) and 50 fg (330 amol), respectively, were found. 

Chao HY, Wang YC, Tang SS, Liu HW. A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A. Toxicon 2004 43(1) 27—34. 

Hanna PA, Jankovic J, Vincent A (1999) Comparison of mouse bioassay and immunoprecipitation assay for botulinum toxin antibodies. J Neurol Neurosurg Psychiatry 66 612-16 Hanson MA, Stevens RC (2000) Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 A resolution. Nat Struct Biol 7 687-92 Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ (2001) The architecture of active zone material at the frog s neuromuscular junction. Nature 409 479-84 Harris JB (1997) Toxic phospholipases in snake venom an introductory review. Symp. zool. Soc. Lond. 70 235-50... 

Unfortimately, there is no simple and readily available assay for botulinum antibodies. The frequency of detectable botulinum antibodies has been found to range from 3% to 5%, with evidence that increased dose and reduced interval between injections are related to the presence of antibodies. Often, the clinician must increase the botulinum toxin dose to maintain the same effect with subsequent treatments. A mean 50% increase in dose may be required for patients with BEB over the first six injections, with no further increase required with later treatments. If a treatment produces an unexpected shorter interval of relief after several good responses from earlier injections, it is likely that the former duration of effect will be reestablished with subsequent treatments. It is possible that the total cumulative toxin dose... 

Like other bacterial ADP-ribosylating toxins (e.g. diphtheria toxin. Pseudomonas aeruginosa exotoxin A, cholera toxin, pertussis toxin, and C. botulinum C2 toxin (Aktories and Just, 1993)), C3 is a mono-ADP-ribosyltransferase (Aktories et ai, 1988b). Treatment of ADP-ribosylated Rho with phosphodiesterase releases 5 -AMP and not phosphoribosyl-AIVtP, a cleavage product of poly(ADP-ribose) (Aktories et ai, 1988b Rubin ef a/., 1988). Accordingly, thymidine, an inhibitor of poly(ADP-ribose)polymerase, does not block C3-like ADP-ribosyltransferases, and can be included in C3 ADP-ribosylation assays to block poly-ADP-ribosylation reactions. 

The full biological activity of C2 toxin, a mixture of components I and II, is obtained by activation of the toxin with trypsin (Miyake and Ohi-shi, 1987 Ohishi et al., 1980 Ohishi et al., 1980 Ohishi, 1987). As described in Section 9.2 (Assay method for the toxin), the full activity of the toxin is produced by a mixture of untrypsinized component I and trypsinized component II. This indicates that activation of the toxin is brought about by the molecular cleavage of component II, but not of component I, by trypsin. Therefore, to study the biological activity of botulinum C2 toxin and the effect of ADP-ribosylation of cytoplasmic actin by C2 toxin on whole cells, it is essential to prepare activated component II (trypsinized component II). 

Sanchez-Prieto J, Shira TS, Evans D, Ashton A, Dolly JO, Nicholls DG (1987) Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes. In Eur. J. Biochem. 165 675-81 Schiavo G, Montecucco C (1995) Tetanus and botulism neurotoxins isolation and assay. Methods Enzymol. 248 643-52... 

Rapid diagnostic assays for detection of toxin agents are available for Botulinum Toxin Clostridium Perfringens Toxin Staphylococcal Enterotoxin B and Staphylococcal Enterotoxins A/C1,2,3/D... 

Sharma, S.K., Ferreira, J.L., Eblen, B.S., and Whiting, R.C. 2006. Detection of type A, B, E, andF Clostridium botulinum neurotoxins in foods by using an amphfied enzyme-hnked immunosorbent assay with digoxigenin-labeled antibodies. Appl. Environ. Micorbiol. 72 1231-1248. 

Rapid diagnostic assays fielded in support Botulinum Toxin of Operation Desert Stonn/Shieldk Clostridium Perfringens Toxin... 

Klewitz, T., Gessler, F., Beer, H., Pflanz, K., and Scheper, T. (2006) Immunochromato-graphic assay for determination of botulinum nenrotoxin type D. Sensors and Actuators B Chemical. 113, 582-589... 

Li, L. and Singh, B.R., Isolation of synaptotagmin as a receptor for types A and E botulinum neurotoxin and analysis of their comparative binding using a new microtiter plate assay, J. Nat. Toxins, 7, 215, 1998. 

Analytical assays would necessarily be more complex and less likely to identify distinct toxins, but they might detect that something unusual was present. Imagine the difficulty of developing a detection system based on molecular weight or other physical characteristics to differentiate among the seven botulinum toxins (molecular weight is the same for all, but each requires a different, specific antibody for identification or therapy). 

Because of the small quantity of toxin protein needed to kill, botulinum toxin exposure does not typically induce an antibody response after exposure. The most likely means of laboratory diagnosis is through enzyme-linked immunosorbent assay identification of botulinum toxin from swabs taken from the nasal mucosa within 24 hours of in-halational exposure. 

Purcell, A. L. and Hoard-Fruchey, H. M. A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes Implications for substrate specificity, Anal. Biochem., 366, 207, 2007. 

The Lawrence Livermore National Laboratory (LLNL) has developed a handheld-type device named Handheld Advanced Nucleic Acid Analyzer (HANAA) (Fig. 3) which is capable of rapid detection of bioagents such as B. anthracis in the field. It uses the TaqMan-based PCR assay system. Due to its small footprint and low weight, it is ideal for field applications. Another development from them is the Nanowire Barcode System which speeds up detection of pathogens such as anthrax, smallpox, ricin, and botulinum. Antibodies of specific pathogens are attached to the nanowires which produce small, reliable, and sensitive detection systems. 

Amperometry at single PC 12 cells has also been used in conjunction with a genetic cell transfection protocol to examine the effects of toxin expression on basal and evoked exocytosis. PC 12 cells have been transfected with the specific endoprotease Botulinum neurotoxin Cl light chain (BoNT/Cl), which cleaves the proteins syntaxin and SNAP-25 [5], The molecular dissection of the mechanisms underlying exocytosis has been motivated by the SNARE hypothesis, which postulates that exocytosis requires the assembly of the plasma membrane proteins syntaxin 1, SNAP-25, and the vesicle associated membrane protein (VAMP) into a complex [5], This SNARE complex then acts as a receptor for cytosolic components of the proposed fusion machinery. Direct evidence for the role of the SNARE proteins in neurotransmission comes from molecular genetic studies in which syntaxin and VAMP have been shown to be required for neurotransmission in Drosophila [47 9] and Caenorhabditis elegans [50,51]. To assess the effects of the disruption of SNARE proteins on exocytosis in PC 12 cells, amperometry has been used in conjunction with a genetic cell transfection assay to establish a... 

CeU-based assay for stability and potency of botulinum neurotoxin type A products In vitro... 

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