{SOMEWHERE OVER THE RAINBOW.}

Saturday, July 21, 2007

title:{Marine biotoxins-detection methods}

Methods of analysis

In general

Ciguatoxins are odourless, tasteless and generally undetectable by any simple test. Therefore, bioassays have traditionally been used to monitor suspected fish. Many native tests for toxicity of fish have been examined including the discolouration of silver coins or copper wire, or the repulsion of flies and ants, but all of these were rejected as invalid (Park, 1994).
Feeding tests to cat or mongoose are simple and relatively sensitive but they are cumbersome and non-quantitative. The mouse bioassay requires purification of fish extracts since the mouse is not very sensitive to ciguatoxin. The alternative mosquito bioassay correlates well with cat and mouse bioassay. Other bioassays that have been developed have used chicken, brine shrimp and guinea pig atrium. All traditional bioassays have one common disadvantage, the lack of specificity for individual toxins. Recent studies have also focused on the development of chemical methods, such as TLC and LC for the detection and quantification of ciguatera-related toxins. Alternative assays based on immunochemical technology have been developed and show greatest promise for use in seafood safety monitoring programmes .

Bioassays

All the mentioned bioassays have the limited chemical specificity for individual toxins in common ,although for a broad screening this property can be advantageous detecting a poisoning. The bioassays are semi-quantitative and sensitive. Ciguatoxin induces characteristic signs of toxicity but the use of some animal species can be problematic in terms of cost and ethical difficulties.
in vivo assays

Mouse bioassay

The mouse bioassay, based on the method described by Banner et al. (1960) is presently the most widely used assay for the detection of ciguatoxins in fish. The method consists of injecting i.p. (intraperitoneal) serially diluted semi-purified or crude toxic extracts into mice and observing the symptoms for 24 hours. This assay has been described for the detection of ciguatoxins in up to 20 mg of ether extract from the flesh of fish. Mice are observed continuously for the first two hours, after that regularly checks are performed. Two mice are tested for each fraction. Mice are housed at 23 ± 2 oC and observed over seven days and signs and times to death recorded. Rectal body temperature is intermittently measured. The relationship between dose and time to death is used to quantify each fraction. Total lethality is expressed in mouse units (MU). For the mix of ciguatoxins found in carnivorous fish (Lewis and Sellin, 1992; Lewis et al. 1991) this relationship is approximated by log MU = 2.3 log (1 + T-1), where MU is the number of mouse units of ciguatoxin injected and T is time to death in hours (see also Table 7.2). One MU is the LD50 dose for a 20 g mouse which is equivalent to 5 ng, 48 ng and 18 ng of CTX-1, CTX-2 en CTX-3, respectively. (Lewis and Sellin, 1992; Lewis et al. 1991). It is recommended that additional purification is undertaken to separate the various toxins, especially the maitotoxins (see Chapter 7.3.1) from ciguatoxins since maitotoxins induce effects in mice, often mistaken for effects of ciguatoxins despite the clear differences (see Table 7.2). Therefore modified extraction procedures have been reported that may improve separation of these two types of toxins (Yokayama et al., 1988; Holmes et al., 1991; Holmes and Lewis, 1994; Legrand et al., 1992).
The mouse assay has been traditionally used but it is unsuitable as a market test. There are other disadvantages such as the variation in mouse weight, that must be limited involving a large breeding colony of the mice, and the death time relationship to dose is non-linear.

Chicken assay

This assay provides a rapid means of assaying the toxicity of fish liver by administering small portions of liver directly into the crop of young chickens at 10 percent of their body weight. Administration of fish flesh is physically more difficult but can be accomplished (Vernoux et al., 1985).

Mongoose and cat assay

For the mongoose (Banner et al., 1960) and cat assay (Lewis, 1987; Bagnis et al., 1985) the same procedure is followed as with chicken, only flesh of fish is fed and also in large quantity (5 to 15 percent of the test animal weight was fed). The cat is less satisfactory as test model because it often regurgitates part of the test meal. Test animals are observed for 48 hours. Although the tests are simple in screening fish for toxicity, they are cumbersome and not quantitative (Bagnis et al., 1987).

Brine shrimp assay

The brine shrimp assay was the first non-vertebrate assay developed. However, false positive results were caused by the toxic effects on brine shrimp of the Tween 80 recommended to emulsify the extract and no toxic effect attributable to ciguatoxin could be detected (Granade et al., 1976; Hungerford, 1993).

Mosquito assay

A bioassay using mosquitoes has also been developed. Only a few laboratories perform this assay, perhaps because of difficulties in obtaining and housing mosquitoes and a lack of familiarity in handling and recognising signs characteristic of intoxication by ciguatoxins. This procedure involves intrathoracic injection of the mosquitoes of serially diluted fish extract, and the toxicity is expressed in mosquito LD50.. It is a rapid assay, depending on a simple extraction requiring a small amount of fish. However, the assay is non-specific and non-quantitative (Bagnis et al., 1985, 1987).

Biochemical assays

Immunoassays

An ideal assay for the detection of marine toxins should be simple, highly sensitive and specific. Therefore the evaluation of marine toxin detection assays has moved in the direction of immunologic analysis (Hokama and Smith, 1990). Immunochemical methods such as a radioimmunoassay (RIA) (Hokama et al., 1977), a competitive enzyme immunoassay (EIA) (Hokama et al., 1983, 1984, 1986), and a rapid enzyme immunoassay stick test (Hokama, 1985; Hokama et al., 1985, 1987) have been developed. Problems with these immunochemical methods are their cross-reactivity with other polyether compounds and the limited antibody supply.

Radioimmunoassay

In 1977, a radioimmunoassay (RIA) was developed for the detection of ciguatoxin directly in contaminated fish (Hokama et al., 1977). In this assay, CTX conjugated to human serum albumin was injected into sheep and rabbits, thereby producing antibodies. The sheep antibody to CTX was used in the RIA after being purified and coupled to 125I as a label. In practice, some false positives were reported. This method could not be used for analyses of large numbers of fishes.
enzyme-linked immunosorbent assay (ELISA)
The practicality of detection improved when Hokama et al. (1983) developed an enzyme immunoassay (EIA) for the detection of CTX. The procedure incorporated a sheep anti-ciguatoxin horseradish peroxidase conjugate and colorimetric determination of absorbance following the enzymatic reaction. The assay was shown to be similar in efficacy to the earlier RIA developed, but less expensive and more practical. However, it was still tedious and therefore abandoned as detection method.

11:27 AM;