Protein and DNA-based assays as complementary tools for fish allergen detection

Abstract

Background: Fish is one of the most important, allergenic foods worldwide. Parvalbumin is the well characterized, major allergen in fish muscle. In this study, we developed a protein- and a DNA-based method for the sensitive detection and authentication of eight commonly consumed fishes in food and compared their applicability. Methods: Fish parvalbumins were purified. Polyclonal, anti-parvalbumin antibodies were raised in rabbits and mice. Protein extracts from food were analyzed by quantitative ELISA. Parvalbumin genes were cloned and sequenced for the design of parvalbumin gene-specific PCR-primers. DNA extracted from food was subjected to specific PCR. Results: Increasing parvalbumin contents were quantified by ELISA in fresh fish, in the order of tuna < mackerel < cod < salmon/trout < redfish < carp < herring. The parvalbumin content of processed fish was up to 67% lower than in fresh fish. In spiked food samples, 1 to 15 ppm fresh fish and 30 to 170 ppm processed fish were still detectable by ELISA. The eight fishes were identified by specific PCR using 0.2 to 10 ng fish DNA. PCRs detected still 3 ppm fresh fish and 30 to 150 ppm processed fish in spiked samples. Conclusions: Both the protein- and the DNA-based method have sufficient sensitivity to protect fish-allergic consumers. The ELISA allows allergen quantification, while the PCR identifies the fish present in the food. The detection limits of both methods vary depending on different factors. Both methods need to be carefully validated for each fish and fish product when used in detection assays.

Cross‐reactivity to fish and chicken meat – a new clinical syndrome

“…Quantitative detection of poultry allergens was modified from a method previously reported . Briefly, authentic food samples (three individual samples each) of raw but also cooked and roasted chicken (breast, leg, and wing) were obtained, followed by protein extraction in triplicate and buffer exchange in phosphate‐buffered saline (pH 7.2).…”

A comprehensive overview of emerging processing techniques and detection methods for seafood allergens

“…Moreover, another inconvenience of the immunoassays is that protein solubility might be altered by processing techniques, which can affect the subsequent detection with protein‐based approaches (Mattison et al., 2016). Instead, other detection methods, such as DNA‐based assays, are necessary for seafood analytic tests since DNA molecules can maintain their integrity better than proteins (A Kuehn et al., 2017).…”

Fish Processing and Digestion Affect Parvalbumins Detectability in Gilthead Seabream and European Seabass

“…Purified antibodies were further biotinylated using the EZ-link™ Sulfo-NHS-LC-Biotinylation kit following manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). ELISA plates (F96 Maxisorp immunoplate, Thermo Fisher Scientific, Waltham, MA, USA) were coated with anti-PV at 500 ng/well overnight at 4 °C as described by Kuehn, Scheuermann, Hilger and Hentges [ 14 ], Kuehn, et al [ 37 ]. Wells were blocked with 300 μL of blocking solution (3% BSA in TBS-0.05% Tween (TBS-T)) for 4 h at room temperature (RT).…”