New mimetic enzymatic sandwich immunoassay system by using oligo(N-isopropylacrylamide) with an active terminal group as a separating support

In this paper, an oligo-N-isopropylacrylamide (ONIP) with a terminal carboxyl group prepared by polymerization of NIP with mercaptoacetic acid was used for conjugating to mouse immunoglobulin G (IgG) via coupling reaction of activated ester with protein amino group. The conjugate not only exhibited a critical temperature of about 32°C, but also maintained higher natural immunological reaction activity of IgG than normal poly-N-isopropylacrylamide-IgG conjugate. These characters were employed to develop a novel polymer-mimetic enzyme sandwich immunoassay method for the determination of goat anti-mouse IgG (anti-IgG) with iron-tetrasulfonatophthalocyanine (FeTSPc) as a new labeling reagent to catalyze the reaction of p-hydroxyphenylacetic acid (p-HPA) and hydrogen peroxide. In the sandwich immunoassay, the anti-IgG first reacted with ONIP-IgG conjugate and then further reacted with FeTSPc-labeled IgG. After the separation process, the (ONIP-IgG)-(anti-IgG)-(IgG-FeTSPc) conjugate moiety served as the catalyst for the fluorogenic reaction of hydrogen peroxide and p-HPA, and thus the anti-IgG could be determined. Under optimum conditions, the calibration graph for the goat anti-mouse IgG was linear over the range of 0.0-1000 ng/ml, with a detection limit of 2.1 ng/ml.