Competitive Enzymatic Fluorescence Immunoassay for Human IgG by Using a Temperature-Sensitive Phase Separating Polymer with Regulated Phase Transition Temperature

摘要

A new enzymatic fluorescence immunoassay for human IgG was developed using a temperature-sensitive polymer, poly(N-isopropylacrylamide-co-acrylamide) [P(NIP-AA)], as a carrier. The lower critical solution temperature of the P(NIP-AA) containing molar fraction of 8% for AA was 37 ℃. In a competitive immunoassay, immobilized IgG and the standard IgG (or sample) competed for binding to a horseradish peroxidase labeled antibody at 33 ℃ in homogeneous format. After changing the temperature to separate the polymer-immune complex, the complex precipitate was re-dissolved and determined by coupling with the fluorescence reaction of hydrogen peroxide and p-hydroxyphenylacetic acid. The calibration graph for human IgG was linear over the range of 100-1000 ng/mL with a detection limit of 2.0 ng/mL. The method is rapid, sensitive and simple. The immune reaction efficiency was improved. In addition, the sensitivity of this method was close to that using traditional microtitration plates as carriers. However, the assay was much faster (the assay time decreased from 100-120 to 30 min). The method has been applied to the determination of the human IgG levels in human sera with satisfactory results.