关键词: 免疫分析, 相分离, 环境敏感高分子, 比较研究, 外膜蛋白

Comparative study of two environment-sensitive polymers in phase separation immunoassay for outer membrane protein (OMP) of Aeromonas hydrophila was carried out. The temperature-sensitive polymer poly(N-isopropylacrylamide) and the pH-sensitive polymer poly(N-isopropylacrylamide-meth- acrylic acid) were synthesized, and conjugated to the anti-OMP antibody (Ab) to form the Ab-polymer complexes with N-hydroxysuccinimidyl acrylate (NAS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as coupling reagents, respectively. In each competitive immunoassay, the standard OMP and the fluorescein isothiocyanate-labeled OMP competed for binding the Ab-polymer in homogeneous format. After changing the external stimuli to separate the polymer-immune complex precipitate, it was re-dissolved and determined by fluorescence method. Under optimum conditions, the calibration graphs for OMP of both systems were linear over the concentration range of 400～3000 ng/mL, with detection limits 84.7 and 39.6 ng/mL, respectively. Compared to temperature-sensitive polymers, pH-sensitive polymers were more suitable to be used as carriers in phase separation immunoassay due to the following advantages. Firstly, the immune process could be conducted at 37 ℃ and therefore the higher reactive efficiency was gotten. Secondly, the pH-sensitive polymer itself contained active groups. The immobilization efficiency and immunological reaction activities of immobilized Ab were improved over the NAS coupling method as antibody was immobilized by the active groups. Moreover, the sensitivity of the immunoassay for OMP using pH-sensitive polymer as a carrier was higher.

Key words: immunoassay, phase separation, environment-sensitive polymer, comparative study, outer membrane protein