Effect Of Matrix Components In Fish On Enzyme-linked Immunosorbent Assay Of Drug Residue

Immunoassays, with the specificity and sensitivity, the relative simplicity andlow-cost, are suitable for routine analysis and screening for large amounts offoodstuffs. A fundenmental problem in immunoassay, however, is that the complexand variable chemical components such as proteins, lipids, carbohydrates and smallmolecules in food samples might greatly interfere with the immunological reactions.The matrix effect is considered a task for further development and application ofimmunoassay of foodstuffs. However, information on the interference from theaquatic products, as well as the mechanism involved, is still very limited. Thus thispaper aims to investigate the most important interferencing components inimmunoassay of drug residue in aquatic products and to explore the exact mechanism.In this study, using three flatfishes (Scophthalmus maximus, Paralichthysolivaceus and Cymoglossus robustus) as samples, the effects of the fish matrix on thecompetitive indirect enzyme-linked immunosorbent assay (ci-ELISA) of antibiotic(norfloxacin) residues were investigated, and the effects of several main componentswere studied. The mechanism of the observed matrix effect was also preliminarilydiscussed. The universal significance of the matrix protein-induced interference to theimmunoassay of food samples containing these main components was explored.Different extraction methods were applied to verify the interferencing matrix.Moreover, the biochemical properties of the two main interferencing proteins inParalichthys olivaceus were determined. The main results are listed as follows:1. Identification and determination of main interferencing components. Withinthe working range of the calibration curves, a significant (P<0.05) but irregularvariation of the inhibition ratio was observed in the presence of fish extracts. Furtherexperiments revealed that such a matrix effect could be caused by some watersolvable fish proteins with a wide range of molecular weight (from lower than14.4 kD to about116.0kD), and the ions from fish muscles may also contribute to theinterference. The changes of pH value and ion strength (0.02mol/L~0.50mol/L)were relevant with the inhibition of ci-ELISA.2. By western blotting and ci-ELISA, the effects of proteins in aquatic sampleson the immunoassay were investigated and evaluated. The result of western blottingindicated that some fish protein components could effectively bind with antibodies,which might resulted in significant loss of the accuracy of the immunoassays. Someprotein components, especially that with molecular weight of35kD~45kD in threeflatfishes and16kD protein in Scophthalmus maximus, were demonstrated strongnon-specific binding ability with different antibodies including IgG and IgE. Furtherexperiments by ci-ELISA revealed that these matrix proteins prefer to interact withenzymes (such as horseradish peroxidase, HRP) labeled antibodies other thanlabel-free ones.3. Such a matrix protein-immunoglobulin interaction was also validated withother aquatic samples including different fishes and shrimps, and similar results wereobserved as those for flatfishes. Different extraction methods were applied to verifythe interferencing matrix and confirmed that the interferencing proteins can easilydissolve out in different solvents, which allowed us to propose a universalsignificance of the protein-induced matrix interference to different immunoassays.4. Two main interferencing proteins (36kD and42kD) in Paralichthys olivaceuswere purified by ammonium sulfate fractional precipitation and ion-exchangechromotagraphy, and the results showed electrophoretic purity. The two proteins wereverified by western blotting and ci-ELISA. The biochemical properties of the twoproteins were briefly summarized as follows: a) the isoelectric point of the suspectedinterfering proteins in flouder was8; b)36kD protein was quite stable at pH3-11while42kD protein was not stable at pH≤5; c) both the proteins were thermal instable,and they were precipitated at60℃; d) they were salt stable. These properties couldoffer instructions to the sample pretreatment process.To summarize, a significant matrix effect was observed in the ci-ELISA, someproteins and ions were demonstrated to contribute to the matrix effect investigated; the non-specific interaction between fish proteins and the IgG was assumed animportant source of the matrix effect in immunoassays and two main interferingproteins was purified and studied. This study fills the research gaps of matrix effect infishery immunoassay and the related mechanism, helps to reduce and avoid theblindness in sample pretreatment, and establishes a theoretical basis for eliminatingthe matrix effect and simplify the pretreatment process.