04 FOOD SAFETY SUPPLEMENT: RAPID METHODS


methodologies are still in the proof of concept phase and are not used widely for food analysis. The most well established immunological methods are the enzyme-linked immuno - sorbent assays (ELISA) and the lateral flow devices (dipstick) that have been commercialised for several types of analytes of interest in food analysis. These assays can be divided into those developed for small molecules such as mycotoxins3 phycotoxins4,5 and those developed for large biomolecules such as allergens6


and . The development of ELISA methods for


large biomolecules, such as food allergens, is a relatively straightforward process where a purified protein extract of the food allergen is


type assays where two antibodies are used in the analysis. These types of assays are typically more sensitive and selective than competitive assays due to the fact that two antibodies need to react. In general, commercial ELISA assays have LOQ values in the range of 1-5 μg/g (ppm) and are adequate for monitoring the food supply in most jurisdictions for allergen cross contamination. Assay development for small molecule


analysis using ELISA techniques is not as straightforward as those for larger proteins, but the principles are the same. Small molecules such as mycotoxins and phycotoxins will not easily produce an immune response on their own and need to be delivered using a carrier


another hapten-protein complex that was developed to contain a different carrier protein than the immunogen. The direct ELISA is quick since only one antibody is used in the competition, but it does require the labelling of this antibody. The indirect method has an extra step in the assay, but the labelled secondary antibodies are commercially available and these assays tend to be more sensitive due to the multiple epitope sites on the primary antibody. Although ELISA methods for large


biomolecules and small molecules have different formats they both rely on the interaction of the analyte with the antibody. This interaction can be very specific and allows for target molecules to be detected and quantified


Figure 1A real time PCR standard curve of our Fusarium test


used as the immunogen to produce polyclonal antibodies that target numerous but specific proteins in the food allergen extract. Although polyclonal antibodies are commonly used for allergen ELISA methods there are some assays that use monoclonal antibodies7,8


that target a


single protein or single epitope on a particular protein. These antibodies have the advantage that the immunological reagents are theoretically limitless and can be regenerated when needed. Whether polyclonal or monoclonal, these assays tend to be sandwich


New Food Volume 14, Issue 5, 2011


protein. There are numerous choices for a carrier protein, but bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) are two commonly used proteins. Once antibodies have been generated towards this hapten-carrier protein complex the ELISA design is typically a direct or indirect competitive assay rather than a sandwich type that is used for larger biomolecules9,10


. In both direct and indirect


ELISA formats, there is a competition for the antibody between the analyte and some analyte complex. This analyte complex is typically


with little preparation of the sample. This has made immunological methods a popular choice for rapid screening for both large and small molecules. Immunological methods have been developed for most of the major allergens6,11 many toxins, including aflatoxins12, ochratoxin13 fusarium toxins2 and phycotoxins5,14


and ,


. These


methods are simple to use, especially the lateral flow methods, but because they rely on the interaction between the analyte and the antibody they are susceptible to anything that could interfere with this interaction. The main


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