Mimetic-enzyme fluorescence immunoassay using a thermal phase separating polymer

Poly-N-isopropylacrylamide (PNIP), a water-soluble, thermally precipitated synthetic polymer, has been conjugated together with a monoclonal antibody and utilized as a novel separation method for an immunoassay. PNIP precipitates out of water above a critical temperature of 31°C, enabling a polymer-bound immune complex to be separated from the solution. These characteristics were used to develop a novel polymer-mimetic enzyme immunoassay method for determination of α-1-fetoprotein (AFP) with hemin as a labeling reagent to catalyze the reaction of p-hydroxyphenyl acetic acid (HPA) and hydrogen peroxide in alkaline medium. After a one-step competitive immunoreaction, the polymer-antibody-antigen-hemin conjugate moiety was determined by coupling the fluorogenic reaction of HPA and hydrogen peroxide. The calibration graph for AFP was linear over the range of 0-380 ng cm^-3 with a detection limit of 1.0 ng cm^-3. This method combines some advantages of both homogeneous and heterogeneous immunoassays, and has been applied to determine AFP in human blood serum with satisfactory results.