Isolation, characterization, and identification of proteins interfering with enzyme-linked immunosorbent assay of antibiotics in fish matrix.

The interaction between some proteins and immune globulin has been confirmed as an important source of matrix interference with the immunoassay of fishery products, but detailed biochemical properties of these proteins have not been indicated. Two interference-inducing proteins (42 and 36 kD) in flounder were isolated, characterized, and identified. Their influences on the immunoassay of norfloxacin were confirmed by western blotting and enzyme-linked immunosorbent assay. The pI value and pH stability of the two proteins were also investigated. Using LC-MS/MS, the two proteins were identified as fructose-bisphosphate aldolase, and such results were partly verified by the aldolase activity of the 42 and 36 kD isolates. Considering the prevalence of these proteins (as multi-functional aldolase of muscles) in foods, these results would help to further understand the matrix effects in various immunoassays as well as the development of effective techniques to improve the efficiency of immunoassays.