Lateral flow assays are a simple and rapid detection method for the qualitative detection of crustacean residues in food, as well as rinse water and environmental samples, including surface swabbing, in production facilities. Real-time testing using on-site lateral flow methods is very important, allowing for quick testing and immediate decision making needed when a fast turnaround or trouble shooting is necessary. Lateral flow tests require few skills and only a minimal amount of training. The detection method is based on an antibody-antigen reaction. In a food sample containing crustacean residues, antigens will bind to antibodies of the test solution. A test strip being soaked into the solution after the binding reaction can be read immediately after a very short incubation time, with one line in the result zone indicating a negative result or two lines a positive result. Lateral flow assays for the detection of crustacean residues in food are rapid, simple, and require no sophisticated procedures or expensive equipment, making them suitable for routine applications, e.g. for monitoring food production lines and equipment in food processing facilities. Though these lateral flow tests are reliable, they are only qualitative, meaning the result shows that either tropomyosin is present (positive, a test line will appear) or tropomyosin cannot be detected (negative, no test line will appear). Thus, lateral flow devices are most often used as quick screening tests for checking the cleanliness of production lines and production equipment, therefore preventing one source of contamination in the final product.
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In a food sample containing crustacean residues, antigens will bind to antibodies of the test solution.
The final product can be screened with a lateral flow device as well, but most often food producers prefer to carry out a quantitative method. Currently, two methods are mainly used for quantitation of crustacean protein in food stuffs: ELISA and PCR.
The ELISA is the main immunological method used for the quantitative detection of allergens in different matrices. For quantitative analysis of shellfish protein, especially tropomyosin, the quantitative sandwich ELISA—a very specific and precise assay—is widely used. Commercial test kits are available, offering limits of detection of around 0.5 to 1 milligram (mg) crustacean protein, depending on the matrix. Wells of a microtiter plate are pre-coated with polyclonal or monoclonal antibodies directed against tropomyosin. An extracted food sample is applied to these wells leading to the binding of tropomyosin to the antibodies. During the next step, the reaction with an enzyme-conjugated secondary antibody directed against tropomyosin leads to a complex formation that can be visualized by the development of a colored reaction product between the particular substrate and the conjugated enzyme on the secondary antibody. As an ELISA easily can be used on a routine basis, demonstrating high precision, it is probably the most utilized method for the detection of food allergens, as well as crustacean residues.
DNA-based methods, as traditional PCR and real-time PCR, detect the genome of the allergenic food as a surrogate for allergenic proteins. Therefore, the extracted DNA is amplified by polymerase chain reaction and detected afterwards (traditional PCR) or simultaneously (real-time PCR).
It must be stated that both methods, ELISA and PCR, have their advantages and disadvantages and that there is no single method fulfilling all needs.
An advantage of PCR is that DNA molecules are more heat stable than proteins, which allows analysis of highly processed foodstuffs. Moreover, DNA extraction is more efficient than protein extraction in difficult food matrices and harsh laboratory extraction conditions can be applied, as DNA is less prone to damage than proteins.
The main disadvantage of PCR is that the protein that causes the allergic reaction is not detected itself. Even if the presence of DNA implied the presence of protein—how could someone convert a DNA copy number to mg allergenic protein? Without knowing the expression level of a certain allergenic protein in a certain matrix, this conversion is almost impossible. For crustacean allergen analysis, normally mitochondrial genes are used as DNA targets as they are present in high copy numbers, resulting in the increased sensitivity of the assay. Again the downside is how someone can convert copy numbers into mg allergenic protein. Currently, only gluten and sulfites are officially regulated by threshold levels stating mg/kilogram concentrations. But how could someone comply, if threshold levels for all the other regulated allergens were introduced?