Botulinum toxins detection

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" ). 

Representatives of medium-size analytes detected by affinity biosensors based on spectroscopy of guided modes include food-safety related analytes such as staphylococcal enterotoxin B , botulinum toxin, and E. coli... 

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. 

R. A. Ogert, J. E. Brown, B. R. Singh, L. C. Shriver-Lake, and F. S. Ligler, Detection ofClostridium botulinum toxin A using a fiber optic-based biosensor. Anal. Biochem. 205(2), 306-312 (1992). 

A major application of LC/ESI/MS is the characterization and detection of toxins, ranging from relatively small molecules, such as mycotoxins and some marine toxins, to the large proteinaceous toxins such as ricin and botulinum toxins. The marine toxin saxitoxin and the plant toxin ricin are specifically listed in Schedule 1 of the CWC as examples of toxins. A comprehensive review of LC/MS in toxin analysis would require a major chapter in its own right. Hancock and D Agostino 1711 reviewed approaches to the mass spectrometric identification of selected low molecular mass toxins. This chapter will describe examples of LC/MS in the analysis of marine, fungal, bacterial, and plant toxins, which are of possible relevance to the CWC. 

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... 

The t-SNARE SNAP-25 is also expressed in the P-cell and has been localized mainly to the plasma membrane (Sadoul etal., 1995). SNAP-25 was cleaved by treatment of SLO-permeabilized cells with botulinum toxins (BoNT) A or E. The two neurotoxins inhibited Ca "-induced insulin exocytosis but failed to abolish the process completely (Sadoul ef al., 1995). This could be due to the failure of the toxins to cleave SNAP-25 already complexed to other fusion proteins (escaping detection by Western blotting) or to the requirement for SNAP-25 at a penultimate step in insulin exocytosis. 

The mouse bioassay is the standard diagnostic laboratory test for botulism (36). The procedure detects whether a type-specific antitoxin protects the mice against any bomlinum toxin that may be present in the clinical specimen. The bioassay, which takes 1-2 days to complete (range 6-96 h), can detect 0.03 ng of botulinum toxin. In addition to the bioassay, anaerobic cultures of clinical specimens can isolate the organism in 7-10 days (range 5-21) days, but a mouse bioassay is necessary to confirm that the culture isolates produced the toxin (36). 

Because terrorist-caused bomlism would most likely be food-borne or inhala-tional, acceptable specimens include feces, gastric aspirate/vomitus, serum, suspected food, and environmental samples (37). Feces, gastric aspirates, or vomitus may be helpful for detecting both food-borne and inhalational botulinum toxin. A walnut-sized, 10-50 g stool sample, placed in a sterile, unbreakable, carefully labeled container, should be sufficient. Enemas are an acceptable alternative for constipated patients. To avoid diluting the toxin and confounding the mouse bioassay, a minimal amount of sterile, nonbacteriostatic water should be used. A 20 ml sample, placed in a sterile, unbreakable, carefully labeled container, should be sufficient. Similarly, 20 ml of gastric aspirate and vomitus, placed in the same type of container, is appropriate. 

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... 

Rivera, V.R., Gamez, F.J., Keener, W.K., White, J.A., and Poli, M.A. (2006) Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead based electrochemiluminescence detec tion. Analytical Biochemistry, 353, 248 256. 

Fig. 9.5.6 Direct detection and amplification of the protein toxin Clostridium botulinum toxin A (toxoid). The upper line is an average of three channels with ant -botulinum antibody on the surface.

The use of photosynthetic enzymes isolated from plants has been implemented in a toxicity monitor (LuminoTox, Lab Bell Inc., Shawinigan, Canada). This system can detect a range of compounds such as hydrocarbons, herbicides, phenols, polycyclic aromatic hydrocarbons (PAHs), and aromatic hydrocarbons. These enzymes have been coupled to screen-printed electrode and have been demonstrated to be able to detect triazine and phenylurea herbicides [79]. Other enzyme inhibitions have been used to detect biotoxins from plant, animals, bacterial, algae, and fungal species (e.g., ricin, botulinum toxins, mycotoxins, cyanobacterial toxins). However, since the identity and specificity of the above toxic compound can be very important during the analysis, other sensor systems such as immunosensors may be preferred to give a better indication to toxin type and identity than the use of enzyme inhibition tests. 

Ahn-Yoon S., DeCory T. R., and Durst R. A., Ganglioside-liposome immunoassay for the detection of botulinum toxin. Anal. Bioanal. Chem., 378(1), 68-75, 2004. 

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). 

The Defence Research Establishment Suffield has initiated a research program to develop methods and equipment for field detection and laboratory identification of proteins and peptides such as Ricin and the Botulinum Toxins. These proteins are difficult to detect and identify even within controlled laboratory settings. In this report, we briefly review capillaiy zone electrophoresis (CZE) and describe a CZE method for the analysis of bio-active peptides. 

Another potential explanation for the imique epidemiology of human botulism was provided in a study of botulinum toxin binding and transcytosis across polarized monolayers of two hiunan colon carcinoma cell lines (T-84 and Caco-2). Substantial binding of iodinated BoNT/A and BoNT/B to hiunan colon carcinoma cells was observed, while minimal binding of type Cl neuro-toxin was detected (Maksymowych and Simpson, 1998). Both type A and B neurotoxins were also efficiently taken up, transcytosed, and released, by the polarized human carcinoma cells, whereas minimal transcytosis of type Cl neurotoxin was observed. The patterns of neurotoxin transcytosis (A and B, but not Cl) observed in these human gut epithelial cell lines correlate with human susceptibility to foodbome botulism (Maksymowych and... 

Gibson, A.M., Modi, N.K., Roberts, T.A., Hambleton, R, and Melling, J., 1988, Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system, J. Appl. Bacteriol. 64 285-291. 

Mestrandrea, L.W, 1974, Rapid detection of Clostridium botulinum toxin by capillary tube diffusion, Appl Microbiol 27 1017-1023. 

Michalik, M., Grzybowski, J., Ligieza, J., and Reiss, J., 1986, Enzyme-linked immunosorbent assay (ELISA) for the detection and differentiation of Clostridium botulinum toxins type A and B, J. Immunol. Meth. 93 225-230. 

Notermans, S., DuFrenne, J., and Van Schothorst, M., 1978, Enzyme-linked immunosorbent assay for detection of Clostridium botulinum toxin type A, Jpn. J. Med. Sci. Biol. 31 81-85. 

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