Development of a Sandwich Enzyme-Linked Immunosorbent Assay for Detection and Quantification of Clam Residues in Food Products

Abstract

Seafood is a frequent cause of allergic reactions to food globally. The presence of undeclared trace amounts of clam can cause allergic reactions in sensitive individuals. Limited tools are available to test food products for the presence of traces of clam. We report on the development of a sandwich ELISA that can detect and quantify clam protein in food. Antisera against a mix of two commercially important clam species, Atlantic Surf (Spisula solidissima) and ocean quahog (Arctica islandica), were raised in rabbit and sheep. A sandwich ELISA was constructed with this antisera, and sensitivity and specificity were evaluated. Also, model food products spiked with clam protein were analyzed to assess the performance of the ELISA. Comparison was made with a commercially available ELISA for crustacea. The lower limit of quantification of the sandwich ELISA is 2.5 ppm clam protein in food samples, allowing the detection of low amounts of clam that may trigger a reaction in clam allergic patients. The sandwich ELISA was highly specific with cross-reactivity only noted for other molluscan shellfish (mussel and scallop). Clam protein in tomato juice and potato cream soup was detected well with recoveries ranging from 65 to 74% and from 74 to 113%, respectively. However when potato cream soup was retorted, the recover fell to 20%, imposing the risk of underestimating the clam content of a food product. A commercially available crustacean ELISA test was not suitable to detect clam protein. The sandwich ELISA described here is suitable for detection and quantification of clam protein in food products. Care should be taken with food products that have been retorted as the results may be underestimated.

Detection of Allergenic Proteins in Foodstuffs: Advantages of the Innovative Multiplex Allergen Microarray-Based Immunoassay Compared to Conventional Methods

“…The sandwich ELISA [ 80 ] is carried out by adsorbing the antigen-specific antibody into the wells. This antibody is generally a specific IgG and will capture the antigen contained in the applied sample [ 81 , 82 ], which can be represented by a food extract. Then, a primary antibody (generally IgG) specific for the searched allergen is added, and it binds to the antigen.…”

Genotyping, physicochemical characterization, and protein isoform quantification of β-casein A2 milk in chinese simmental and Angus cattle

“…The optical density values of the samples detected from a sandwich ELISA assay are related to the positive interaction between β-CN A2 protein isoform and the ELISA's enzyme-labeled antibodies, as well as the relative comparison of this interaction to the protein concentration in the standard curve. Other studies have found that the sandwich ELISA assay is the most reliable test for IgE-mediated allergenic substances in food, such as casein, albumin, globulin, gliadin, gluten, and clam proteins, with high precision, sensitivity, and relative ease of use (Bernard et al, 2021;Bosman et al, 2021;Koppelman et al, 2021;Madrid et al, 2021). Herein, the higher concentration of β-CN A2 protein isoform in Angus is likely linked with the larger plasma concentration of proline residue present in milk.…”

“…In an attempt to in-vitro quantify β-CN A2 protein isoform concentration in order to obtain insights into its bioactive potential, the sandwich ELISA assay at optical density OD 450 wavelength was effectively performed in this study and did not cross-react with β-CN A1 protein. The detection using the sandwich ELISA assay allows a more direct reference of the presence of protein residues (Koppelman et al, 2021). In this assay, β-CN A2 protein isoform can be determined by the colorimetric reaction after the antigen binding with the particular enzyme-labeled antibody.…”