Analytical Sciences

Surface Plasmon Resonance Immunosensor for IgE Analysis Using Two Types of Anti-IgE Antibodies with Different Active Recognition Sites

Yan LI, Jujie REN, Hizuru NAKAJIMA, Nobuaki SOH, Koji NAKANO, and Toshihiko IMATO

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi, Fukuoka 819-0395, Japan

A simple and novel method for the determination of an IgE antibody based on a surface plasmon resonance immunosensor for the diagnosis of an allergy is described. The method involves the use of an anti-IgE(D) antibody and an anti-IgE(H) antibody, which reacts with the Ce2 domain and the Ce3 domain of the IgE antibody. The anti-IgE(D) antibody was immobilized on the gold surface of a sensor chip by physical adsorption. An IgE antibody sample was incubated by adding it to an anti-IgE(H) antibody solution to form an anti-IgE(H) immunocomplex through a reaction of the Ce3 domain of the IgE antibody. The incubated solution was introduced onto the sensor chip and the immunocomplex of the IgE-anti-IgE(H) then reacted with the anti-IgE(D) antibody immobilized on the sensor chip through the Ce2 domain of the IgE antibody part of the IgE-anti-IgE(H) immunocomplex. The detection limit of the present method for the determination of the IgE antibody was about 10 ppb. The affinity constants for the anti-IgE(H) antibody immunocomplex with the IgE antibody in solution and that of the anti-IgE(H) antibody immunocomplex with the IgE antibody immobilized on the sensor chip by a biotin-streptavidin interaction were estimated to be 4.1 × 10⁷ M^-1 and 5.8 × 10⁶ M^-1, respectively. The affinity constant for the immunocomplex of the anti-IgE(H) antibody with the IgE antibody with the anti-IgE(D) immobilized on the sensor chip was estimated to be 4.9 × 10⁷ M^-1, 20-times larger than the affinity constant for the IgE antibody immunocomplex with the anti-IgE(D) antibody immobilized on the sensor chip, based on a direct immunoassay method of the IgE antibody under the same experimental conditions.

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