Nowadays, seafood plays a very important role in human nutrition worldwide. It is among the major foods consumed globally, mainly due to a large movement towards healthier eating. International trade in seafood and new seafood products is growing rapidly and there are more and more reports of adverse reactions in the population consuming seafood. The generic term “crustaceans” includes all arthropodic aquatic animals, with six major subgroups and 44,000 species. Hidden crustacean proteins in food represent a critical problem for people with crustacean allergies. Sufferers have to avoid the consumption of food containing crustaceans very strictly. Still, cross-contamination can occur as a consequence of the production process.
International organizations, such as the Codex Alimentarius Commission of the World Health Organization, or WHO, and the Food and Agriculture Organization, or FAO, have discussed a labeling system for allergenic ingredients in processed food and labeling is now recommended for eight food groups, including crustaceans. In the European Union, there are different regulations that establish labeling must be present in regards to the main types of food allergens to warn consumers. In the U.S., the Food Allergen Labeling and Consumer Protection Act of 2004 includes the eight major allergen groups—namely milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, wheat, and soybeans. However, allergens can be present in the final food product even though the ingredients are not allergens due to cross-contamination between raw materials, equipment, or production lines. Therefore, there is increasing need for sensitive detection of crustacean residues in foodstuffs, as well as diagnosis and treatment of seafood allergies to protect the allergic consumer and to ensure supervision of labeling requirements by the authorities responsible.
Food allergies in general have become an important health problem and represent a food safety issue. Food allergies, defined as an adverse immune response to food proteins that the human body mistakenly identifies as harmful, affect approximately 5 to 8 percent of children and 2 to 3 percent of adults in developed countries, with rising prevalence. A true allergic reaction is defined as a type-one hypersensitivity activating a specific type of white blood cells, the mast cells, leading to an immunoglobulin E response. Histamine and inflammatory mediators, such as cytokines, are released, leading to different symptoms, including itchiness, gastrointestinal disorders, dyspnea, or even anaphylaxis and death.
Crustaceans are among the eight food groups thought to lead to allergies triggered by immunoglobulin E antibodies worldwide. Hypersensitivity reactions to different ingested crustaceans, including shrimps, crabs, lobsters, and others are among the most frequent causes of food allergic reactions. Furthermore, crustaceans are the third most important foodstuffs inducing food-related anaphylaxis. Several studies have shown that even minute amounts of ingested seafood allergens can trigger very quick allergic reactions. It is assumed that even inhaled airborne allergens can lead to sensitization and allergic reactions. Very commonly, crustacean allergies appear at later stages of life and the likeliness of them being outgrown, as it is very often the case in childhood allergies, is very small.
The major allergen in shrimps and other crustaceans is the ubiquitous muscle protein tropomyosin, which is responsible for ingestion-related allergic reactions. It is a highly conserved protein, is homologous in different species, and shows a considerable rate of identicalness. Its high cross-reactivity even to insects like house dust mites means tropomyosin is considered as a possible cause of cross-reactivity between food and respiratory allergens of animal origins. There are other allergens that have been identified in crustaceans in the past few years, including arginine kinase and myosin, but they have not yet been characterized completely. In contrast to other seafood allergens, tropomyosin seems to be relatively resistant to acidic digestion and heat. The heat stability makes this protein suitable for the analysis of crustacean residues in processed food samples. Therefore, allergen-specific detection assays in food products are available for crustacean tropomyosin.
The detection methods for food allergens, including crustacean protein, have to be very specific and must detect minute quantities of allergen in very complex food matrices. At present, there are qualitative and quantitative test methods for crustacean residues available. Few techniques detecting crustacean protein, actually tropomyosin, are available, including immunological methods based on a specific antigen-antibody reaction, such as lateral flow assays and enzyme-linked immunosorbent assays (ELISA), as well as DNA-based methods such as polymerase-chain reaction (PCR) technologies, mainly real-time PCR.
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.
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?
In general, testing issues are mainly represented by the strongly varying matrices that have to be tested (matrix effect). The detection limit of any test, therefore, will vary depending on the sample matrix to be analyzed. Although providers of test kits often have a validation protocol to be submitted with the test kit upon request, laboratories using commercial test kits must establish in-house controls on detection limits and quantification limits in actual matrices. As there are no reference methods and no certified reference materials for allergens available, one has to keep in mind that results are not necessarily comparable between different test systems. Accredited methods should be used in official controls. The National Food Agency is accredited for the analysis of shellfish protein in foodstuffs.
Kuster is the product manager at Romer Labs. Reach her at firstname.lastname@example.org.