Fluorescence immunoassay system based on the use of a pH-sensitive phase-separating polymer

Poly(N-isopropylacrylamide-co-methacrylic acid) [P(NIPAAm-co-MAA)], a linea r water-soluble pH-sensitive phase-separating polymer, was synthesized and used as a novel separation carrier for the reactants in immunoassay. This p olymer precipitates out of water below a critical pH 5.8 at 37 degreesC and redissolves when the pH of solution is above 6.2. The characteristic of th is polymer makes it possible to carry out the Immunochemical steps of an im munoassay in a true solution and then to quickly separate the resulting pro duct from the reaction mixture. The above approach was applied to determina tion of a-fetoprotein with the competitive immunoassay format. Compared wit h traditional ELISA using the same reactants, the proposed method was much faster (the assay time decreased from 100-120 to 30 min) and showed similar sensitivity, i.e., 0.04 ng/mL. In addition, a sandwich immunoassay method for the determination of hepatitis B surface antigen was also studied, and the results showed that the pH phase-separating immunoassay could be carrie d out through a sandwich or a competitive method. This general technique ma y also be used for a wide variety of separation processes in addition to im munoassay, in which a specific component is to be isolated for analysis, re covery, or disposal. (C) 2001 Academic Press.