Competitive Enzyme-Linked Immunosorbent Assay for Quantitative Detection of Fish Proteins in Heat-Processed Crab Meat

According to the Food Allergen Labeling and Consumer Protection Act, fish is one of the eight major allergen foods. Reliable analytical methods for the detection of food ingredients derived from fish are important for the protection of fish allergic individuals. Surimi is defined as a protein concentrate made of fish muscle and is usually used for the manufacture of shellfish analogues or substitutes. They are typically made from white-flesh fish (e.g. pollock or whiting) and may be a hidden ingredient in some processed foods. This study developed a monoclonal antibody (MAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) for quantitative detection of fish protein in heat-processed crab meat. A previously developed MAb 8F5, which recognizes a 36-kDa thermostable fish protein, was used in this study. The icELISA was optimized and 11 sets of spiked samples adulterated at 0.01% to 5% were used to produce sets of standard inhibition curves and transformed linear curves for quantification of surimi in crab samples. The effect of cooking methods and cooking times on the detectability of a representative surimi product was also examined. The 50% inhibitory concentration values (IC50) of the icELISA were 4.0% (g/g)-10.0% (g/g) for surimi samples and 3.9% (g/g) for fish samples. Fish samples have smaller IC50 value than surimi samples, which means that fish samples contain a larger amount of antigenic fish proteins than surimi samples at the same total protein concentration. The difference in IC50 also indicated that the fish samples have a higher sensitivity than surimi products analyzed by this icELISA. This assay exhibited a linear response within a wide concentration range (0.01 to 100%) of all 11 sets of spiked samples. It can reliably distinguish fish products from crab meat and the detection limit for these spiked samples was less than 5.0% (g/g). This assay exhibited low intra-assay variability (< 6.3%) and inter-assay variability (< 4.1%). The different effects of heat treatments on the detectability of a representative surimi product indicated that accurate quantification of fish proteins in crab meat requires a standard curve obtained from matched sample matrix with matched cooking methods and time. This developed immunoassay demonstrated its usefulness for the detection and quantification of low levels of fish ingredient in processed crab meat, and would help discourage the illegal practice of substituting crab meat by cheaper surimi products at the retail and restaurant levels.