Development of a sensitive sandwich ELISA with broad species specificity for improved fish allergen detection

A sensitive Enzyme-linked Immunosorbent Assay (ELISA) with improved broad species specificity was developed for the detection of southern hemisphere fish residues in processed foods. The polyclonal antibodies were raised against parvalbumins from 13 fish species representing 7 fish orders selected for their molecular diversity and immunoreactivity profile. The optimized ELISA-2 (based on the rabbit capture antibody (RB#4) - sheep detection antibody (S2#4) pair) displayed an improved detection limit of 0.6 μg/L (3.7 μg of /kg). Our immunoreactivity-directed species selection approach in the strategized antibody production significantly improved the detection of no or weakly immunoreactive fish species previously not detected immunochemically. Of 37 commercially important fish species tested, the ELISA-2 could detect 28 fish species (76%). The optimized sample extraction with a buffer additive achieved good protein recoveries of 87.2 - 117.3% (within the AOAC recommended range). The ELISA-2 was able to detect fish residues in five highly processed food products.