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

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.

SUBMITTER: Kochanowski M

PROVIDER: S-EPMC7598195 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Kochanowski Maciej M Różycki Mirosław M Dąbrowska Joanna J Karamon Jacek J Sroka Jacek J Antolak Ewelina E Bełcik Aneta A Cencek Tomasz T

Pathogens (Basel, Switzerland) 20200923 10

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 ...[more]

PMID: 32977528

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Project description:BackgroundSerine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP).MethodsThe full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted.ResultsThe AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity.ConclusionsOur findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site.

Dataset Information

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

Publications

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

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