Immunochemical methods have been used in diagnosis of diseases both for humans and animals for many years. However, wide application of these methods for analytical purpose was not initiated until the nineteen fifties when Yalow developed a radioimmunoassy for insulin¹⁾. In addition to the specific antibody, the modern immunochemical methods use a sensitive marker for the analyte and a good method to separate the bound and free analyte in the specific antigen (Ag) and antibody (Ab) interaction. The assay systems became a new highly sensitive, specific and simplified versatile tool for the analysis of many biologically active substances, including low molecular contaminants such as mycotoxin. In the last four decades, new approaches to make sensitive markers have been made. For example, by conjugating the enzymes to the Ag or Ab to amplify the signal, more sensitive and versatile enzyme-immunoassay systems have been developed. With the availability of sensitive instruments, more fluorescence-tagged markers are now being used. Simplified and effective methods for the separation of free and bound species are now available. In addition to using animals for Ab production, monoclonal Ab technology was introduced. Such developments have led to a wide application of immunoassays for the analysis of contaminants in foods and agricultural products. Recent development of novel separation techniques and sensitive detection systems have led to the development of several immunochemical based biosensors. Thus, a new dimension of immunoassay system is now emerged. Whereas many types of immunoassays are now available for mycotoxins, most approaches are based on the competition of binding between unlabeled toxin in the sample and labeled toxin in the assay system for the specific binding sites of Ab molecules. Because of limited space, this review will focus only on important developments and applications of immunoassays for mycotoxins. For details, the readers should consult the original papers and recent reviews both on the overall analytical methods^2-9) and immunoassays for mycotoxins^10-18).

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The extract of Talaromyces flavus IFM52668 showed the characteristic antifungal activity against Aspergillus fumigatus. In the course of a search for the active compounds, funicone, vermistatin, and a new funicone derivative, 9,14-epoxy-11-deoxyfunicone, were isolated along with emerin. The structure of 9,14-epoxy-11-deoxyfunicone was determined from the analysis of the spectroscopic investigation. The antifungal activity of the compounds was discussed.

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The possibility that rubratoxin B induces apoptosis in hepatoma cells was investigated. Exposure to rubratoxin B caused the shrinkage of HuH-7 cells, and this effect was dosedependent. In 40 μg/ml rubratoxin B-treated HepG2 and HuH-7 cells, we observed condensation of chromatin and the presence of dense spherical structures in the nuclei, showing that rubratoxin B induces apoptosis in hepatoma cells. DNA fragmentation detected by modified TdT-mediated dUTP nick end labeling (TUNEL) method was also increased in a dosedependent manner in HepG2 cells, and a similar trend was observed in HuH-7 cells. These results indicated that rubratoxin B-induced apoptosis in HepG2 and HuH-7 cells is accompanied by DNA fragmentation.

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The effects of anthocyanins on hepatotoxin rubratoxin B-treated HL60 cells were investigated. Anthocyanins are known to have an anti-oxidant activity and corresponding protective effects on hepatic injury. While cyanidin 3-O-β-_D-glucoside (Cy 3-glc) and peonidin 3-O-β-_D-glucoside (Pn 3-glc) slightly impaired the inhibitory effect of rubratoxin B on cell proliferation, their effects were not significant. In contrast, malvidin 3-O-β-_D-glucoside (Mv 3-glc) drastically strengthened the effect of rubratoxin B. Structural difference between Mv 3- glc and the others is the presence of an R group in the 5' position at the B ring, suggesting that the group in this position affected the proliferation of rubratoxin B-treated cells. The results for cytokine secretion were apparently different from those for cell proliferation. The effects of the three anthocyanins on rubratoxin B-induced secretion of interleukin-8 and tumor necrosis factor-α were not substantial. At 100 μM, all the anthocyanins markedly boosted the secretion of monocyte chemotactic protein (MCP)-1, indicating that the common structure shared by the three anthocyanins affected rubratoxin B-induced MCP-1 secretion. In this study, we thus found that anthocyanins exert their activities in ways that either do or do not depend on the identity of the R group in the 5' position at their B ring.

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Trends in analytical methods used in mycotoxin analyses over the last 25 years are discussed based on results from WHO and FAPAS^®. In 1978, most determinations of aflatoxin B₁ in maize samples were done utilizing TLC methods. The percentage of TLC methods decreased to 48 % in 1989 and to 7 % in 2002. Alternatively, the use of HPLC has increased from less than 5 % in 1978 to 36 % in 1989 and 77 % in 2002. ELISA based methods have also been used in more recent tests, but the percentage of laboratories using ELISA is not high. The percentage of data falling within |z| ≤ 2 increased steadily from 50 % in 1978 to 82 % in 2002. Similar trends over time were observed for aflatoxin B₁ in peanut, aflatoxin M₁ in milk and ochratoxin A data.

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[in Japanese]

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