Determination of outer membrane protein of Aeromonas hydrophila by pH controlled phase separation fluoroimmunoassay

A fluorescence immunoassay was developed for outer membrane protein (OMP) of Aeromonas hydrophila using a pH-sensitive polymer as a carrier. The pH-sensitive polymer was conjugated to the anti-OMP antibody with N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI). In a sandwich immunoassay, OMP first reacted with the anti-OMP antibody immobilized on pH-sensitive polymer and then further reacted with fluorescein isothiocyanate (FITC)-labeled the anti-OMP antibody in 37 degrees C in homogeneous format. After changing the pH to separate the polymer-immune complex precipitate, it was re-dissolved and determined by fluorescence method. The calibration graph for OMP was linear in the range of 0.4-30 mg/L with a detection limit of 31 mu g/L. The method is rapid, sensitive and simple. The immobilization efficiency, immunological reaction activities of immobilized antibody were improved as antibody was immobilized via reaction of activated carboxylic group by EDCI with protein amino group.