Effect of storage and processing on the immunodetectability of fish proteins using pooled monoclonal antibodies in ELISA and dot blot

Abstract

Fish are listed as one of the “big-eight” major allergenic food groups. A previously developed indirect enzyme-linked immunosorbent assay (iELISA) based on three pooled monoclonal antibodies (mAbs, including 2G9, 2F3 and 8F5) reliably can detect 63 commercially important fish species without cross reactivity with non-fish proteins. However, fish are often stored frozen or processed before preparation for consumption, both of which can induce structural changes in the muscle proteins that may affect the immunoreactivity and thus the detectability of the target fish protein in an immunoassay. In this study, we investigated the effects of the length of frozen storage (up to 11 months at −20 °C), cooking time (boiling 0–8 min), and processing method (smoking and salting) on the detectability of selected fish samples with two immunoassays, iELISA and dot blot, using the three pooled mAbs. The results from both immunoassays showed that all stored fish tissue and protein extracts could be detected after frozen storage for months after purchase. More decrease in immunoreactivity was observed in stored protein extracts than in the extracts prepared from stored fish tissue. Cooking increased the immunoreactivity of the fish samples tested. Although all the salted and smoked fish samples were detected by dot blot, not all were detected by iELISA. These findings confirm the effectiveness of these pooled mAb-based assays for rapid detection of fish protein in a variety of frozen and processed products.