MIMETIC-ENZYME FLUORESCENCE IMMUNOASSAY USING A THERMAL PHASE-SEPARATING POLYMER

Poly-N-isopropylacrylamide (PNIP), a water-soluble, thermally precipit ated 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 de grees C, enabling a polymer-bound immune complex to be separated from the solution. These characteristics were used to develop a novel polym er-mimetic enzyme immunoassay method for determination of alpha-1-feto protein (AFP) with hemin as a labeling reagent to catalyze the reactio n of p-hydroxyphenyl acetic acid (HPA) and hydrogen peroxide in alkali ne medium. After a one-step competitive immunoreaction, the polymer-an tibody-antigen-hemin conjugate moiety was determined by coupling the f luorogenic reaction of HPA and hydrogen peroxide, The calibration grap h for AFP was linear over the range of 0-380 ng cm(-3) with a detectio n limit of 1.0 ng cm(-3). This method combines some advantages of both homogeneous and heterogeneous immunoassays, and has been applied to d etermine AFP in human blood serum with satisfactory results.