Mimetic-enzyme fluorescence immunoassay using a thermal phase separating polymer

Abstract

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.