Botulinum neurotoxins assay

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... 

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. 

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. 

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. 

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... 

Doellgast, G.J., Beard, G.A., Bottoms, J.D., Cheng, T., Roh, B.H., Roman, M.G., Hall, P.A., and Triscott, M.X., 1994, Enzyme-linked immunosorbent assay and enzyme-linked coagulation assay for detection of Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes with dual-label antibodies, J. Clin. Microbiol 32 105-111. 

Notermans, S., and Nagel, J., Assays for botulinum and tetanus toxins, in Botulinum Neurotoxin and Tetanus Toxin, Simpson, L.L., ed.. Academic Press, Inc., San Diego (1989). 

Until recently, there have been only two primary techniques available for the detection of botulinum neurotoxins. The first of these, which is the most widely accepted and sensitive technique for the detection of botulinum neurotoxins in semm and food extracts, is the mouse bioassay (Sakaguchi, 1983). Although the mouse bioassay is the most sensitive method, with the ability to detect less than 5 mouse 50% lethal doses (MLD5os)/mL, the assay takes up to four days to complete and requires a large number of mice if the toxin is to be quantified. In addition, the mouse toxicity results are not in themselves specific specificity is imparted only by carrying out parallel toxin neutralization tests with homologous antisera (Shone et al., 1985). Furthermore, as future modifications are made to botulinum neurotoxins relative to their use as therapeutic agents, quantification relative to its toxicity to mice may not be possible. Thus, despite the apparent sensitivity offered by the mouse bioassay, its use as a routine detection technique for botulinum neurotoxins is not only impractical, but also may be obsolete in some areas of research. 

Furthermore, research efforts described herein, have resulted in at least a ten-fold improvement in the detection of Clostridium botulinum neurotoxin using a fiberoptic immunosensor. Additionally, the increased sensitivity of the assay by using affinity purified polyclonal antibodies as both the immobilized capture antibodies and the fluorescently labeled antibodies suggests that the investigation into additional assay systems may be worthwhile. 

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... 

Ransom, G.M., Lee, W.Fl., Elliot, E.L., and Lattuada, C.P., Enzyme-linked immunosorbent assays (ELISAs) to detect botulinum toxins using high titer rabbit antisera, in Botulinum and Tetanus Neurotoxins, DasGupta, B.R., ed.. Plenum Press, New York (1993). 

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