Enterotoxin, detection methods

Immunological methods Methods such as gel-agglutination tests were introduced many years ago (according to Outcherlony) however they are not sufficiently sensitive. Reversed passive latex agglutination kits (SET-RPLA and TST-RPLA) are presently applied for enterotoxin detection and are more sensitive than gel diffusion (Wieneke, 1988). The ELISA technique is also very popular - toxins are detected via VIDAS STAPH ENTEROTOXIN SET and indirect double sandwich ELISA (Meyrand et al., 1998). 

Source Ler, S.G., Lee, F.K., Gopalakrishnakone, P. (2006) Trends in detection of warfare agents detection methods for ricin, staphylococcal enterotoxin B and T-2 toxin. Journal of Chromatography A, 1133,1-12. 

Sensitive Detection Methods. Detection of toxin production by use of the more sensitive detection methods such as reversed passive latex agglutination (RPLA) (Igarashi et al., 1986) and enzyme-linked immunosorbent assay (ELISA) indicated that some strains produced only 10 to 20 ng/ml, which was not detectable by the OSP method. This was confirmed when 100-fold concentrates of culture supernatant fluids from five strains that tested positive for SEA by ELISA were positive by the OSP method. Examination of 110 strains that were positive when administered to monkeys, but were negative for any of the identified enterotoxins by MOA-OSP, were examined for enterotoxin production by ELISA (Kokan and Bergdoll, 1987). Twenty-six strains were found to be positive with most of them producing enterotoxin D (SED) (Table 7). Some of the strains were isolated from food poisoning outbreaks. The amount of toxin produced appeared to be 10-15 ng/ml of culture supernatant fluid. 

It can be noted that the production of toxins is not one of the properties on which the classifications are based, as production of the toxins cuts across species, even to include an occasional CNS. Production of the toxins by any of the CNS is considered rare, if indeed actual however, there is one report that coagulase-negative staphylococci from several different species do produce one or more of the identified toxins (Valle et al.,1990). This is a debatable question because one of the sensitive detection methods, ELISA, was used to assay for toxin production. The amount of toxin produced by these strains was less than 10 ng/ml of culture supernatant fluid. The fact that the ELISA methods are sensitive to less than 1 ng/ml of culture supernatant fluid, it is questionable whether a strain producing less than 10 ng/ml can be considered toxin-positive. It is an open question as to how much toxin a strain should produce to be labeled a toxin producer, as this has not been defined. Some of the strains are under study to determine if the toxin production reported can be verified and whether these strains can produce enterotoxin in foods. 

A third type of ELISA method for enterotoxin detection in foods makes use of dip sticks that have wells with nitrocellulose paper in the bottoms containing the specific antibodies to the enterotoxins, each well containing an antibody to a different enterotoxin (Fig. 3). Only one test is necessary per sample because all of the enterotoxins can be tested for with the same dip stick (Transia, 8 Rue Saint Jean de Dieu, 69007 Lyon, France). The monoclonal antibodies used were developed in the Bergdoll laboratory (Thompson, et al., 1984 Thompson et al.,1986). The sensitivity of the method was less than that of the ball method and showed non-specific reaction with the SEC detection (Wieneke, 1991 Table 12). The nonspecific reaction may have been due to the use of polyclonal antibody for the second SEC antibody. 

The extraction method is relatively simple as usually no concentration is necessary for the sensitive detection methods (Freed et al.,1982 Table 14). Usually only about 50% of the enterotoxin is recoverable from the foods (Fig. 4). It is necessary to centrifuge the food extracts at relatively high speed which may be a problem for some laboratories, particularly those in the developing countries. This could be a particular problem if the RPLA method is used because the extract must be completely clear to avoid interference with the agglutination. In this case, it is necessary for some extracts to be filtered through a non-protein adsorbing filter. Preparation of the food extracts for use in the ball method... 

The current procedures are adequate for detection of enterotoxin in foods and it is possible to do them in one day. There is always the request that the time for doing an analysis be shortened, but in reality, there is no great need to obtain results in less than the time now required. We have come a long way in improving the methods for enterotoxin detection in foods, but it is likely that further improvements will be made in the future. 

Three groups of methods have been applied to the detection of staphylococcal enterotoxins ... 

In one report, an array of antigens patterned on a substrate was constructed for detection of IgG antibodies from various species. This method, termed miniaturized mosaic immunoassay, is rendered possible by using the PDMS channel for patterning materials [1027]. In another report, various antibodies were patterned via the PDMS channel on a substrate for detection of three antigens FI antigen (from Yersinia pestis), staphylococcal enterotoxin B, and D-dimer (DDi, a marker of sepsis and thrombotic disorder) [1028],... 

Medina, M.B. 2003. Detection of staphylococcal enterotoxin B (SEB) with surface plasmon resonance biosensor. J Rapid Methods Autom Microbiol 11 225-243. 

The staphylococcal toxin must be separated from food constituents and concentrated to detect trace amounts. The toxin is then identified by specific precipitation with antiserum as follows (1) the selective adsorption of the enterotoxin from an extract of the food onto ion exchange resins and (2) the use of physical and chemical procedures for the selective removal of food constituents leaving the enterotoxin in solution. More recently rapid methods based on monoclonal antibodies (e.g., enzyme-linked immunosorbent assay, reverse passive latex agglutination) have been developed for detecting very low levels of enterotoxin in food. 

K8. Kobayashi, K., Seto, K., Ueguchi, M., Makino, M., Ishibashi, M., Akasaka, S., and Yamamoto, K., A new simple method of detecting cholera enterotoxin gene from Vibrio cholerae 01 isolates, lgaku noAyumi 150, 509-510 (1989). 

Dolman CE, Wilson RJ, Cockroff WH A new method of detecting staphylococcal enterotoxin. Can J Public Health 1936 27 489-493. 

Azaspiracid poisoning (AZP) is the most recently discovered of the toxic syndromes from shellfish consumption. The symptoms of acute AZP intoxication closely resembles those associated with diarrhetic shellfish poisoning (DSP), and include diarrhea, vomiting, and headache and the first confirmed incident was in 1995 (Netherlands), following the consumption of mussels (Mytilus edulis) that were cultivated in Ireland (Satake et al., 1998). Since these symptoms are similar to bacterial enterotoxin poisoning, and analytical methods for the detection of these toxins have only been developed in recent years, it is probable that there have been many unreported cases of this poisoning. The first azaspiracid to be identified was AZAl (Figure 35.1, = H R = CH3),... 

Vernozy-Rozand C., Mazuy-Cruchaudet C., Bavai C., and Richard Y., Comparison of three immunological methods for detecting staphylococcal enterotoxins from food, Lett. Appl. Microbiol., 39(6), 490-494, 2004. 

Biological samples from patients are generally not as useful for diagnosis of intoxications as they are for diagnosis of infectious diseases or chemical intoxications. The same is true of postmortem samples. Ricin can be identified with immunoassays in extracts of lung, liver, stomach, and intestines up to 24 hours after aerosol exposure. High doses of ricin can be identified in fixed lung tissue of aerosol-exposed laboratory animals by immunohistochemical methods. The staphylococcal enterotoxins can be detected by immunoassay in bronchial washes. Like blood and swab samples, postmortem tissue or fluid samples should be kept cold, preferably frozen, until they can be assayed. 

Kijek TM, Rossi CA, Moss D, Parker RW, Henchal EA (2000) Rapid and sensitive immunomagnetic-electrochemiluminescent detection of staphyloccocal enterotoxin B. J Immunol Methods 236(l-2) 9-17... 

Other assays have concerned the immobilization of the antibody antialpha-fetoprotein on a silicon structure Au/Si/SiOa using glutaraldehyde. The interaction with the antigen in solution gives rise to variations in capacitance, which are measured at a potential difference of +4 V/SCE [249], and give a detection limit of 1 ng/ml. The method can also be applied to the detection of staphylococcus enterotoxin B by immobilizing the monoclonal antibody anti-enterotoxin (IgG) on the silica surface of the Si/Si02 heterostructure [250]. 

Giugliano, L. G., Stringer, M. R, and Drasar, B. S., 1983, Detection of Clostridium perfringens enterotoxin by tissue culture and double-gel diffusion methods, J. Med. Microbiol. 16 233-237. 

All methods for detection of the enterotoxins are based on the use of antibodies prepared against the enterotoxins. Most of the antibodies in use have been prepared in rabbits using the individual purified enterotoxins (Robbins Bergdoll, 1984). These polyclonal antibodies react with the enterotoxins in gels to give precipitin reactions which are highly... 

Gel Diffusion Methods. Many types of gel reactions have been used in the detection of the enterotoxins, the most common ones being some form of either the Ouchterlony gel plate or the microslide. These methods have been used widely in the determination of the enterotoxigenicity of staphylococcal strains. The modification of the Ouchterlony gel plate test that is used in the Food Research Institute and recommended to others is the optimum sensitivity plate (OSP) method (Robbins et al., 1974 Fig. 2). It is easy to use and in conjunction with production of the enterotoxins by the membrane-over-agar or sac culture methods (Robbins et al.,1974) is of adequate sensitivity for detection of most enterotoxigenic... 

Detection of enterotoxin in foods requires sensitive methods, such as the ELISA and RPLA methods. The quantity of enterotoxin present in foods involved in food poisoning outbreaks may vary considerably, from less than 1 ng/g to greater than 50 ng/g. Usually, little difficulty is encountered in detecting enterotoxin in foods involved in food poisoning outbreaks, however, outbreaks do occur in which the amount of enterotoxin is less than 1 ng/g, such as the case with the chocolate milk (Table 8). In such cases, the enterotoxin can be detected only by the most sensitive methods. Another situation in which it is essential to use a very sensitive method is in determining the safety of a food for consumption. In this situation it is necessary to show that no enterotoxin is present by the most sensitive methods available. 

The ELISA methods were applied to the detection of the enterotoxins in foods soon after they were originally developed for the detection of other proteins. Essentially all ELISA methods are of the sandwich type in which the antibody is treated with the unknown sample before the antibody-enterotoxin complex is treated with the enzyme-antibody conjugate. This type is preferred because the amount of enzyme and, thus, the color developed from the enzyme-substrate reaction, is directly proportional to the amount of enterotoxin present in the unknown sample. This eliminates the need for the highly purified enterotoxins as crude or only partially purified enterotoxin is needed for preparation of a standard curve. 

An RPLA kit produced in Japan is available commercially through Oxoid (Oxoid Limited, Wade Road, Basingstoke, Hants RG24 OOW, England). The method is adequately sensitive for the detection of enterotoxin in most foods that are implicated in food poisoning outbreaks (Igarashi, et al., 1985 Table 13) however, it may be inadequate for detection of the small amounts of enterotoxin that sometimes is present (Table 8). This method is the sensitive method of choice for the examination of staphylococcal strains for the production of enterotoxin. 

Wieneke, A. A., and Gilbert, R. J. 1987. Comparison of four methods for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning. Int. J. Food Microbiol. 4 135-143. 

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