Affinity-based detection of mycotoxins has relied mainly on the use of antibodies or fragments
thereof. However, recently DNA-based aptam- ers and molecularly imprinted polymers (MIP) have been developed and applied for the specific analysis of mycotoxins (Maragos et al., 2009b). Aflatoxin B: (Ma et al., 2014), aflatoxin M: (Mal- hotra et al., 2014), DON (Eifler, 2014), Fumonisin B: (McKeague et al., 2010), OTA (Cruz-Aguado and Penner., 2008), T2-toxin (Chen et al., 2014) and zearalenone (Chen et al., 2013) can now be detected on the basis of single-stranded DNA oli- go nucleotide aptamers of 50-100 nucleotides in length which form a three-dimensional structure specifically upon contact with the analyte molecule (Sampson, 2003). MIP form hollow three-dimensional structures in which the target molecule can be trapped specifically by mutual electrostatic interaction of side groups (Haupt and Mosbach, 2000). They provide solid-phase materials with a molecular memory for the target compounds and have been utilized in solid-phase extraction of and sensors for the detection of fumonisin analogues (de Smet et al., 2009), DON and zearalenone (Weiss et al., 2003), OTA (Baggiani et al., 2001) and moniliformin (Appell et al., 2007).
Antibody-based immunochemical detection methods are based on the stereo-specific binding reaction between the variable domain of an immunoglobulin (antibody) and its antigen. Extensive information about development and use of immunoglobulins is available in specialized literature and in textbooks (Owen et al., 2013; Murphy, 2012; Delves et al., 2011; Subramanian, 2004; Campbell, 2000).
Various immunochemical assays have been described for the detection of fungal mycelia. However, the largest part of the literature covers detection of clinically relevant species. A smaller number of publications deals with the analysis of phytopathogenic fungi and other plant-associated species. The following literature review describes immunochemical assays that have been set up for the analysis of fungi that have been described to grow on cereals or cereal-based foods. The monoclonal antibodies and polyclonal antisera described have not necessarily been described for analysis of barley and wheat or malt produced therefrom but the methods used can in principle be used for that purpose after proper modification of the sample preparation protocol. Notermans and Heuvelman (1985) were the first to develop specific antisera for the detection of foodborne moulds. Their antisera were raised by immunization of rabbits with freeze- dried preparations of protein precipitates from cultures of Mucor racemosus, Fusarium oxysporum, and Penicillium verrucosum. The enzyme-linked immunosorbent assay (ELISA) assay set up with the P. verrucosum specific antiserum was shown to detect most Penicillium species (Notermans et al., 1986). Kamphuis et al. (1989) developed an immunochemical latex agglutination assay for the rapid detection of a broad spectrum of foodborne Aspergillus and Penicillium species. Later, Dewey et al. (1990) used antibodies specific for Penicil- lium islandicum to set up an immunological test strip format for the analysis of rice samples. Tsai and Cousin (1990) published an ELISA for the simultaneous detection of different moulds such as Aspergillus versicolor, Cladosporium herbarum, Geotrichum candidum, Mucor circinelloides and Peni- cillium chrysogenum in yoghurt and cheese. An even wider range of grain-associated fungi, including Penicillium spp. and Aspergillus spp. as well as typical field fungi such as Septoria spp. and Fusarium spp., can be detected with the assays described by Banks and colleagues (Banks et al., 1994, 1996). Penicillium aurantiogriseum was detected in cereals by Lu et al. (1994) in a highly specific manner using a monoclonal antiserum against the fungus. Chang and Yu (1997) developed a rapid immunochemical detection method for Aspergillus parasiticus and Penicillium citrinum and applied it to the analysis of rice and maize. Tsai and Yu (1999) used the same antiserum for the analysis of cereals. The detection of mycelia of aflatoxin-producing fungi in cereals and foodstuffs using an immunochemical method was described by several authors (Shapira et al., 1997; Yong and Cousin, 2001). Immunochemical methods have also been developed for the detection of Fusarium contaminations in cereals. A polyclonal serum for the collective determination of F. avenaceum, F. culmorum and F. graminearum contamination was obtained after immunization with the supernatant of a still culture of F. culmorum by Beyer et al. (1993). An even broader spectrum of detected Fusarium species was reported by Iyer and Cousin (2003), who applied the assay to the analysis of grains and food. Rohde and Rabenstein (2005) reported similar results with polyclonal antisera produced in rabbits against mycelia of F. gramine- arum and F. culmorum, respectively, which they applied for the analysis of wheat grains. Banks et al. (1996) described the use of a monoclonal antibody for the specific detection of F. avenaceum in cereals. The use of polyclonal antibodies raised in chicken eggs for the detection of F. poae was described by Gan et al. (1997). In an attempt to detect infection of maize with fumonisin-producing Fusarium spp., Meirelles et al. (2006) set up an ELISA-based immunoassay using a polyclonal antiserum raised against an unknown peptide from a F. verticillioides culture. Finally, Meyer and Dewey (2000) used a culture supernatant to raise monoclonal antibodies for the detection of Botrytis cinerea in a wide variety of plants.
Even more importance has been given to the immunochemical analysis of mycotoxins as harmful fungal secondary metabolites in brewing cereals and malt. Zheng et al. (2006), Maragos (2006) and Maragos and Busman (2010) provide comprehensive reviews of the literature describing classical and modern methods of immunochemical mycotoxin analysis. Assays based on ELISA have been described for the detection of a great variety of different mycotoxins, including those described previously in section, ‘Mycotoxins. Those assays are commercially available for application in brewing cereals, malt, and beer after appropriate sample preparation (Rahmani et al., 2009). This type of assay is based on a competitive reaction between free and solid-phase-bound mycotoxin for binding to an enzyme-labelled specific antibody. Usually reactions are performed in 96-well microtitre plates as the solid phase. Following specific binding, unbound analyte and antibodies are removed by washing and detection of the binding event is performed after addition of a chromogenic enzyme substrate (Turner et al., 2009). The developed colour is measured spectrophotometrically and light absorption is inversely proportional to analyte concentration. The major advantages of ELISA are speed, low-cost and user-friendliness, because it is portable and easy to perform even under on-site conditions in the brewery (Pleadin et al., 2012). Numerous variations of the ELISA format have been described, differing mainly in enzyme labels and substrates used and in the choice of the solid phase-bound reaction partner.
Other variations of immunoassays involve the use of fluorochrome-labelled mycotoxins or antibodies enabling direct signal detection.
Fluorescence polarization immunoassays do not involve solid phase binding of components. They have been set up for many important mycotoxins. The method measures the change in fluorescence intensity in a solution upon binding of a fluorescently labelled antigen to a specific antibody in relation to the concentration of free antigen in a sample, thus allowing its direct quantification with high sensitivity within minutes (Maragos, 2009a). All major mycotoxins potentially occurring in raw materials and beer can be detected with this method even though no application has been described so far for that specific purpose.
Another variation of the immunosorbent assay is a technology in which the specific antibody or the antigen is immobilized on the surface of a membrane and brought into contact with the free antigen in a sample solution. The method is marketed as a lateral flow device (LFD) assay and is available for the detection of all important mycotoxins, including those occurring in the barley-to-beer chain (Anfossi et al., 2013). Indirect and direct assay formats provide the carrier-bound mycotoxin or the mycotoxin-specific antibody, respectively, immobilized on a nylon membrane. Upon sample application, the free mycotoxin binds to a specific antibody bound to gold nano particles (GNP) in the indirect format or is just mixed with a GNP- bound conjugate of the toxin in direct format. As the sample fluid moves along the LFD membrane by capillary force, hitherto unreacted antibodies are retained by binding to the immobilized antigen in the indirect assay or free and GNP-bound toxin molecules compete for binding to the immobilized antibody in direct assays. In both cases the immobilized GNP result in formation of a red-coloured line, the intensity of which is inversely proportional to the concentration of free mycotoxin in a sample. The same principle has recently been used for the parallel analysis of aflatoxins, DON, and zearale- none in a multiplex assay (Song et al., 2014).
Apart from the aforementioned mycotoxin assays, in which the affinity-based binding event is transduced visually, mostly as a colour change, several other principles of biosensoric signal transduction have been applied to the study of mycotoxins (Pohanka et al., 2007). Electrochemical transducers measure electron movement (potentio- metric), current change (amperometric) or changes in conductivity (conductometric) occurring due to the binding event or due to activity of a label enzyme. Optical biosensors use optical phenomena occurring due to binding of affinity molecules such as antibodies or aptamers to a glass surface or a gold-coated glass surface, the light reflective properties of which are influenced by the binding event. Surface plasmon resonance (SPR)-based sensors have been used to detect and quantify the most important mycotoxins occurring in food (Li et al., 2012). Fibreoptic or optical waveguide biosensors make use of the induction of an evanescent light wave when light is totally reflected at the inner surface of a glass fibre. Provided the evanescent wave has got the right wavelength, it can be absorbed by fluorophore molecules in close proximity to the surface of the fibre and induce fluorescence, which can be measured by coupling back into the fibre (Maragos and Thompson, 1999). Biosenors based on the technology have been developed for afla- toxins and fumonisins (Thompson and Maragos, 1996; Maragos and Thompson, 1999).