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Comparison of Immobilization Modes in pH‐Sensitive Phase Separation Immunoassay
Author(s) -
Lin Peng,
Guo Songlin,
Wang Yilei,
Wang Weigang,
Chen Jinming,
Jia Xiwei,
Wang Guodong
Publication year - 2009
Publication title -
chinese journal of chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.28
H-Index - 41
eISSN - 1614-7065
pISSN - 1001-604X
DOI - 10.1002/cjoc.200990367
Subject(s) - chemistry , carbodiimide , horseradish peroxidase , immunoassay , chromatography , hydrogen peroxide , peroxidase , linear range , detection limit , enzyme , antibody , polymer chemistry , organic chemistry , immunology , biology
Three immobilization modes of antigen to the polymers in the pH‐sensitive phase separation immunoassay were investigated and compared. The results showed that the immobilization mode in the presence of N ‐ethyl‐ N′ ‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDCI) rendered the most desirable results. The immobilization efficiencies and immunological reaction activities of immobilized antigen of this mode were improved over the other two modes. The novel immobilization mode by EDCI was used in the pH‐sensitive phase separation immunoassay for rabbit IgG (Ag). In the competitive immunoassay, immobilized Ag and the standard Ag (or sample) competed for binding to a horseradish peroxidase labeled antibody at 37°C in a homogeneous format. After changing the pH to separate the polymer‐immune complex, the complex precipitate was re‐dissolved and determined by coupling with the color reaction of hydrogen peroxide and o ‐phenylenediamine. The linear range of this determination was between 100–1400 ng/mL. Compared to the traditional enzyme‐linked immunosorbent assays (ELISA) using the same reactants, the proposed method was quiet fast (the time decreased from 100−120 to 30 min) and showed similar sensitivity, i.e ., 6.0 ng/mL.