A novel polymer-mimetic enzyme immunoassay system based on thermal phase separating technique

Poly-N-isopropylacrylamide (PNIP), a water-soluble, thermally precipitating synthetic polymer, has been conjugated with a monoclonal mouse anti-human α-1-fetoprotein (AFP) antibody and utilized in a novel separating method for an immunoassay. The PNIP polymer precipitates from water above a critical temperature of about 31°C, resulting in the immune complex bound to PNIP to be separated from the solution. These characteristics were used to develop a novel polymer-mimetic enzyme-linked immunoassay system for the determination of AFP with hemin (horseradish peroxidase substitute) as a labeling reagent to catalyze the reaction of thiamine and hydrogen peroxide at pH 8.5. In a competitive immunoassay, it utilized the fast homogeneous antigen-antibody immune complexation reaction and a simple heterogeneous separation process. After the one-step immunoreaction, the polymer-antibody-antigen-hemin conjugate moiety was determined by measuring the fluorescence produced in a solution containing thiamine and hydrogen peroxide. The calibration graph for AFP was linear over the range 0-450ng/ml with a detection limit of 1.2ng/ml. The method is rapid, simple, sensitive and inexpensive. In addition, a solid-phase sandwich immunoassay method for the determination of hepatitis B surface antigen (HBsAg) was also proposed, and the results show that the mimetic enzyme immunoassay is possible by both the sandwich method and the competitive method. Copyright (C) 1998 Elsevier Science B.V.