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

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 0–450 ng/ml with a detection limit of 1.2 ng/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.