A NOVEL POLYMER-MIMETIC ENZYME-IMMUNOASSAY SYSTEM BASED ON THERMAL PHASE-SEPARATING TECHNIQUE

Poly-N-isopropylacrylamide (PNIP), a water-soluble, thermally precipit ating synthetic polymer, has been conjugated with a monoclonal mouse a nti-human a-l-fetoprotein (AFP) antibody and utilized in a novel separ ating method for an immunoassay. The PNIP polymer precipitates from wa ter above a critical temperature of about 31 degrees C, resulting in t he immune complex bound to PNIP to be separated from the solution. The se characteristics were used to develop a novel polymer-mimetic enzyme -linked immunoassay system for the determination of AFP with hemin (ho rseradish peroxidase substitute) as a labeling reagent to catalyze the reaction of thiamine and hydrogen peroxide at pH 8.5. In a competitiv e immunoassay, it utilized the fast homogeneous antigen-antibody immun e complexation reaction and a simple heterogeneous separation process. After the onestep immunoreaction, the polymer-antibody-antigen-hemin conjugate moiety was determined by measuring the fluorescence produced in a solution containing thiamine and hydrogen peroxide. The calibrat ion graph for AFP was linear over the range 0-450 ng/ml with a detecti on limit of 1.2 ng/ml. The method is rapid, simple, sensitive and inex pensive. In addition, a solid-phase sandwich immunoassay method for th e determination of hepatitis B surface antigen (HBsAg) was also propos ed, and the results show that the mimetic enzyme immunoassay is possib le by both the sandwich method and the competitive method. (C) 1998 El sevier Science B.V. All rights reserved.