A quantitative sandwich ELISA for the detection of Anisakis simplex protein in seafood

Abstract

There is a growing awareness by consumers and food safety authorities regarding the possible presence of parasites or parasite-related potentially hazardous substances in seafood. Anisakis simplex is among the most frequently occurring parasites in wild-caught marine fish. Except for various visual inspection techniques and PCRbased methods for the detection of more or less intact worms or parasite DNA, respectively, there are at present no validated methods for the quantification of A. simplex proteins in processed fish products. This work describes the development and validation of a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification and analysis of proteins from A. simplex in seafood products. The ELISA is based on a polyclonal rabbit anti-A. simplex antibody for capture and a biotinylated conjugate of the same antibody for detection. The ELISA is specific for A. simplex and does not cross-react with other species. Recoveries ranged from 72-101% in typical food matrixes, while intra-and inter-assay precisions were \11 and \25%, respectively. With a limit of detection of 1.1 lg A. simplex protein/g of sample, the sensitivity of the A. simplex sandwich ELISA appears to be sufficient to detect even low levels in seafood products.

Quantitative indirect ELISA for determination of walnut proteins in foods

“…A good correlation is evident between protein concentrations and optical density values in the range of 25 to 800 ng/mL (correlation coefficient R 2 >0.99). A quantitative sandwich ELISA for detection of Anisakis simplex protein in seafood has been reported with a determination range of the standard curve between 10 and 125 ng/ mL (30), narrower than the range of the indirect ELISA in this study.…”

Laboratory diagnostic methods

“…The detection of the zoonotic species of anisakids in fresh and processed fish products has been successful at detecting as little as 1-40 pmol anisakid DNA in 25-30 g fish using real-time PCR (Fang et al, 2011;Lopez and Pardo, 2010) and shows promise in replacing AD techniques. These immunodiagnostic assays have reported detection rates of as little as one larva in 100 g of fish meat (Werner et al, 2011) and a recovery rate of 82.5% for A. simplex allergens (Rodríguez-Mahillo et al, 2010). These immunodiagnostic assays have reported detection rates of as little as one larva in 100 g of fish meat (Werner et al, 2011) and a recovery rate of 82.5% for A. simplex allergens (Rodríguez-Mahillo et al, 2010).…”

“…Genetic analysis of Anisakis simplex complex, the principal causative agent of anisakiasis, comprising Anisakis pegreffii, A. simplex sensu stricto, and A. simplex C can be accomplished by analyses of the ribosomal ITS data, the mitochondrial cytochrome c oxidase 2 gene as well as allozyme electrophoretic data (Mattiucci et al, 2011;Arizono et al, 2012). ELISAs for the detection of crude proteins (Werner et al, 2011), or more specifically the primary heatstable allergen Ani s 4 of A. simplex (Rodríguez-Mahillo et al, 2010) in fish muscle have also been developed as alternatives to conventional testing. ELISAs for the detection of crude proteins (Werner et al, 2011), or more specifically the primary heatstable allergen Ani s 4 of A. simplex (Rodríguez-Mahillo et al, 2010) in fish muscle have also been developed as alternatives to conventional testing.…”

Methods for Anisakis simplex detection in fish and fishery productsMethods for Anisakis simplex detection in fish and fishery products

“…Ze względu na wspomnianą oporność kutikuli Anisakis na dezintegrację, efektywność homogenizacji wzmacnia użycie sonikacji. Po homogenizacji próbki odbywa się ekstrakcja alergenów A. simplex, która jest przeprowadzana w buforach, głównie fosforanowym (PBS) lub Tris-HCL (TBS), o pH około 7 (35,(44)(45)(46). Po procesie ekstrakcji próbka badana jest testem ELISA.…”