Characterization of a 36 Kda Fish Protein and Its Application to the Development of an Immunoassay for the Detection of Fish Muscle

Fish is classed as a major allergenic food under the Food Allergen Labeling and Consumer Protection Act (FALCPA), which requires accurate information on food allergens to be included in the label of food products. Fishmeal, a fish by-product, is a common protein source in animal feed but it has been banned for use in ruminant feed under the transmissible spongiform encephalopathy regulations in European Union, Japan and Australia. Currently, there is no convenient and reliable method available for the detection of fish in food and animal feed. The overall goal of this study was, therefore, to develop a sandwich enzyme linked immunosorbent assay (sELISA) for the detection of fish and fish products. The specific objectives were to: 1) identify a suitable marker protein to indicate the presence of fish in food; and 2) develop an immunoassay based on this marker protein for the detection of fish in foods. A 36 kDa thermal-stable protein recognized by a previously developed fish-specific monoclonal antibody (MAb) 8F5 in cooked fish extract was present in all 55 fish species tested. Because of its presence in common food fish species and the thermal-stability of the 36 kDa protein, this was identified as a potentially suitable marker protein. The 36 kDa protein was further characterized along with fish tropomyosin because of their similarity in molecular weight, protein banding pattern and thermal-stability. The results showed that the 36 kDa protein and fish tropomyosin both exhibited molecular migration in urea gel and were recognized by MAb 8F5 in an immunoblot test. In addition, the 36 kDa protein and fish tropomyosin had matching amino acid compositions and identical protein sequence (12 amino acid residues). Furthermore, the MAb 8F5 epitope contained the conserved region of fish tropomyosin. Based on these results, the 36 kDa was verified to be fish tropomyosin, a ubiquitous muscle protein with equal distribution in muscle tissue in different locations. Fish tropomyosin was therefore deemed a suitable marker protein for fish detection in an immunoassay. In order to develop a user-friendly sELISA for fish detection, a polyclonal antibody (PAb) was raised against fish tropomyosin to pair with MAb 8F5. However, PAb competes with MAb 8F5 for the same epitope, so PAb was used as both the capture antibody and the detection antibody for the sELISA after depleting the non-specific cross-reactivity. The optimized assay was specific to both raw and cooked samples of all 64 fish species tested with no cross-reaction with shellfish, land animals and food additives. The assay was also capable of detecting fishmeal and processed fish products (salting, smoking, canning). The assay exhibited low intra- (%CV ≤ 8.9%) and inter-assay variability (%CV ≤ 9.4%). The limit of detection of the assay was 0.1 ppm for both raw and cooked fish (pollock and basa) in crab meat. The polyclonal antibody-based sELISA developed for this study is expected to be a useful tool for the qualitative detection of fish muscle protein in food products to reduce the risks associated with fish allergies and enforce the food safety laws.