Toxins detection

The detection and quantification of cyanobacterial toxins quoted in the above examples required methods which have been undergoing rapid development in recent years, and as the need for greater understanding of the properties and occurrence of the toxins continues to grow, these are continuing to be developed. This has resulted in methods of cyanobacterial toxin detection which are more sensitive, quantitative, reliable, specific and humane. Many of these methods are presented and discussed in the proceedings of a recent conference. 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

Not all cyanobacterial blooms and scums contain detectable levels of toxins. Indeed, the incidence of toxicity detection by mouse bioassay, and toxin detection by HPLC among environmental samples, ranges from about 40% to However, in view of this high occurrence, it is the policy of regulatory authorities and water supply operators in some countries to assume that blooms of cyanobacteria are toxic until tested and found to be otherwise. In the absence of available analytical facilities or expertise or for logistical reasons, this precautionary principle should be regarded as sensible and prudent. 

Some Formal Considerations. As with most natural toxins, detection methods for the saxitoxins are an essential prerequisite for most studies of them, as well as for monitoring programs to ensure the safety of food products that may contain them. Furthermore, the degree of success of such efforts is dependant on the characteristics of the detection method used. Detection of the saxitoxins is particularly challenging because of the large number of different but related compounds that must be dealt with, the low levels that must be detected, and their chemical characteristics. Given these factors it is useful to dwell briefly on some underlying principles. 

The PSP toxins represent a real challenge to the analytical chemist interested in developing a method for their detection. There are a great variety of closely related toxin structures (Figure 1) and the need exists to determine the level of each individually. They are totally non-volatile and lack any useful UV absorption. These characteristics coupled with the very low levels found in most samples (sub-ppm) eliminates most traditional chromatographic techniques such as GC and HPLC with UVA S detection. However, by the conversion of the toxins to fluorescent derivatives (J), the problem of detection of the toxins is solved. It has been found that the fluorescent technique is highly sensitive and specific for PSP toxins and many of the current analytical methods for the toxins utilize fluorescent detection. With the toxin detection problem solved, the development of a useful HPLC method was possible and somewhat straightforward. 

The application of the fluorescence derivatization technique in an HPLC method involves utilization of a post column reaction system (PCRS) as shown in Figure 3 to carry out the wet chemistry involved. The reaction is a 2-step process with oxidation of the toxins by periodate at pH 7.8 followed by acidification with nitric acid. Among the factors that influence toxin detection in the PCRS are periodate concentration, oxidation pH, oxidation temperature, reaction time, and final pH. By far, the most important of these factors is oxidation pH and, unfortunately, there is not one set of reaction conditions that is optimum for all of the PSP toxins. The reaction conditions outlined in Table I, while not optimized for any particular toxin, were developed to allow for adequate detection of all of the toxins involved. Care must be exercised in setting up an HPLC for the PSP toxins to duplicate the conditions as closely as possible to those specified in order to achieve consistent adequate detection limits. 

Mass Spectrometry. Mass spectrometry holds great promise for low-level toxin detection. Previous studies employed electron impact (El), desorption chemical ionization (DCI), fast atom bombardment (FAB), and cesium ion liquid secondary ion mass spectrometry (LSIMS) to generate positive or negative ion mass spectra (15-17, 21-23). Firm detection limits have yet to be reported for the brevetoxins. Preliminary results from our laboratory demonstrated that levels as low as 500 ng PbTx-2 or PbTx-3 were detected by using ammonia DCI and scans of 500-1000 amu (unpublished data). We expect significant improvement by manipulation of the DCI conditions and selected monitoring of the molecular ion or the ammonia adduction. 

Fang Y., Fratos A.G., Lahiri J., Ganglioside microarrays for toxin detection, Langmuir 19 1500-1505,2003. 

Ogert R.A., Shriver-Lake L.C., Ligler F.S., Toxin detection using a fiber optic-based biosensor, Proc. SPIE. 1885 11-17,1993. 

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" ). 

Steven R. Tannenbaum, a member of the Institute of Medicine, has a Ph.D. in food science and technology from the Massachusetts Institute of Technology, where he is currently the codirector and Underwood-Prescott Professor, Division of Bioengineering and Environmental Health, and professor of chemistry, Department of Chemistry. Dr. Tannenbaum s research interests include the chemistry and pathophysiology of nitric oxide, the quantitative measurement of human exposure to carcinogens, and tissue-based microsensors for toxin detection and drug metabolism. He has been a member of the NRC Board on Environmental Studies and Toxicology and has served on several NRC committees. 

Acetylcholinesterase inhibition has been widely used for pesticide detection [88-94], but less exploited than protein phosphatase inhibition for cyanobacterial toxin detection. Nevertheless, the anatoxin-a(s) has more inhibition power than most insecticides, as demonstrated by the higher inhibition rates [95]. In order to detect toxin concentrations smaller than usually, mutant enzymes with increased sensitivity were obtained by genetic engineering strategies residue replacement, deletion, insertion and combination of mutations. Modifications close to the active site, located at the bottom of a narrow gorge, made the entrance of the toxin easier and enhanced the sensitivity of the enzyme. 

A partially purified Bacillus thurlnglensis var. israelensls (Bti) 6-endotoxin was used to Immunize rabbits. The antisera obtained have an improved specificity towards the mosquito larvacidal activity of the toxin, as opposed to antiserum raised when the whole crystal was used as immunogen. Using a two step/indirect ELISA (enzyme linked immunosorbent assay) procedure developed in our laboratory, fourteen experimental formulations were tested, and the results were compared with bioassays. An average of 69.1 international units 20% c.v. was found to associate with each ug of toxin detected by the ELISA. Our data indicate that when toxin specific antisera are available, Immunoassays can be used to predict the biological activity of Bti samples with reasonable accuracy. 

In terms of biosensing applications using such layers, again cholera toxin detection on a porous silicon substrate [85] has been reported. Also biotin-avidin interaction by QCM [86], glutamate detection [87], as well as protein membrane interactions [88, 89] have been studied. 

Unfortunately, the dose-survival times for the DSP toxins in the mouse assay fluctuate considerably and fatty acids interfere with the assay, giving false-positive results consequently, a suckling mouse assay that has been developed and used for control of DSP measures fluid accumulation after injection of the shellfish extract. Considerable effort has been applied recently to development of chemical assays to replace these bioassays. As a result a good high performance liquid chromatography (HPLC) procedure has been developed to identify individual PSP toxins (detection limit for saxitoxin = 20 fg per 100 g of meats 0.2 ppm), an excellent HPLC procedure (detection limit for okadaic acid = 400 ngg 0.4 ppm), a commercially available immunoassay (detection limit for okadaic acid=lfg per 100 g of meats 0.01 ppm) for DSP, and a totally satisfactory HPLC procedure for ASP (detection limit for domoic acid = 750 ngg 0.75 ppm). 

Currently the pCCA devices are being investigated for applications in cancer therapy, selection of drug resistance modulators, environmental toxin detection, and so forth. The CCA technique is being commercialized by the Hurel Corp. 

Kamp, L., Church, J. and Rubio, F., 2008. Development of sensitive Immunoessay formats for algal toxin detection, 6th National Monitoring Conference, Atlantic City, New lersey. 

Rucker, V.C., Havenstrite, K.L. and Herr, A.E., 2005. Antibody microarrays for native toxin detection. Anal. Biochem., 339, pp. 262-270. 

If both assays are measuring the toxic element of the 6-endotoxin, numbers in the last column should be identical for all 14 formulations. An average of 69.3 14.9 IU was found to be associated with each microgram of toxin detected with the ELISA procedure. 

Sundaram P V 1990 Waveguide for T2 toxin detection using quartz-immobilized... 

---