Development and Application of Novel Chemiluminescence Immunoassays for Highly Sensitive Detection of Anisakis simplex Proteins in Thermally Processed Seafood.

Maciej Kochanowski,Jacek Sroka,Joanna Dąbrowska,Aneta Bełcik,Tomasz Cencek,Ewelina Antolak,Jacek Karamon,Mirosław Różycki

doi:10.3390/pathogens9100777

Maciej Kochanowski, Jacek Sroka + Show 6 more

Open Access

https://doi.org/10.3390/pathogens9100777

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Abstract

The third-stage larvae (L3) of Anisakis simplex are the most important source of hidden allergens in seafood products. However, there exist no commercial methods for detecting Anisakis proteins in food. Furthermore, only a few methods have been validated for the detection of A. simplex in thermally processed food. The aims of our study are (i) the development and validation of high-sensitivity chemiluminescent (CL) immunoassays for the detection of A. simplex proteins in processed seafood, (ii) and A. simplex antigen detection in common seafood products from Polish markets. We developed and validated CL sandwich ELISA (S-ELISA) and CL competitive ELISA (C-ELISA) methods for A. simplex proteins detection in food, with respective detection limits of 0.5 and 5 ng/mL. The usefulness of the assays for detecting A. simplex proteins in highly processed food was evaluated by examination of autoclaved canned fish spiked with A. simplex larvae (1–8 larvae/200 g). Commercial real-time PCR was unable to detect A. simplex in autoclaved samples at all levels of enrichment with Anisakis larvae. CL-S-ELISA was used to test various types of seafood products from Polish markets. Among all tested products (n = 259), 28% were positive. A. simplex antigens were found mostly (n = 39) in smoked fish products: mackerel, herring, cod, and hake. Other positive samples were found in marinated herrings, canned cod livers, canned mackerels, and surimi sticks. In tuna, Atlantic argentine, anchovy, sardine, sprat, and squid products, A. simplex antigens were not detected. This study provides novel effective tools for the detection of A. simplex proteins in processed food and highlights the potential allergic hazards for Anisakis-sensitized Polish consumers of seafood.

Highlights

Anisakis simplex is a pathogenic nematode belonging to the genus Anisakis, family Anisakidae, and order Ascaridida
As CL-sandwich ELISA (S-ELISA) showed the Results of the examination seafood products fromproducts
We address two issues related to A. simplex: (i) we developed and validated two effective CL-ELISA methods for detecting A. simplex proteins in processed foods; (ii) and we used

Summary

Introduction

Anisakis simplex is a pathogenic nematode belonging to the genus Anisakis, family Anisakidae, and order Ascaridida. A. simplex is a nematode widespread in many different species of marine organisms, mainly in the Arctic-Boreal region [1,2,3,4,5,6]. Humans can become accidental hosts as a result of the consumption of live third-stage larvae in seafood dishes. In humans, this disease is called anisakiasis, which mainly affects the Pathogens 2020, 9, 777; doi:10.3390/pathogens9100777 www.mdpi.com/journal/pathogens. In addition to the pathogenic effect associated with tissue damage, Anisakis larvae can cause allergic reactions in sensitized humans. Many Anisakis allergens are resistant to low or high temperatures and pepsin digestion; dead larvae—even those subjected to intensive technological processes such as autoclaving—may cause hypersensitivity reactions [7]. Cases of Anisakis-induced hypersensitivity by contact with and inhalation of allergens have been reported [8]

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