Analytical Sciences

Single-Step Sandwich Immunoreaction in a Square Glass Capillary Immobilizing Capture and Enzyme-linked Antibodies for Simplified Enzyme-linked Immunosorbent Assay

Erina TSUTSUMI, Terence G. HENARES, Shun-ichi FUNANO, Kunio KAWAMURA, Tatsuro ENDO, and Hideaki HISAMOTO

Graduate School of Engineering, Osaka Prefecture University, Osaka 599–8531, Japan

To simplify the complicated operation steps and to minimize sample and reagent amounts for enzyme-linked immunosorbent assays (ELISA), we developed a square glass capillary immunosensor containing both covalently immobilized capture antibodies and physically adsorbed enzyme-linked antibodies. The immobilization of capture antibodies (anti-human IgG) was carried out by the treatment of 3-aminopropyltriethoxy silane, glutaraldehyde, and protein-A, followed by affinity capture of the antibody. In contrast, the enzyme-linked antibodies (alkaline phosphatase (ALP)-linked anti-human IgG) were physically adsorbed on the four corners of the capillary with the aid of polyethylene glycol (PEG) acting as a scaffold. A nanoliter volume of antigen (human IgG)-containing sample solution was introduced via capillary action. This addition resulted in the release and diffusion of ALP-linked anti-human IgG into the bulk solution. This event led to a 20-min single-step sandwich immunoreaction at the inner wall of capillary; the reaction was detected through the reaction with fluorescein diphosphate (FDP) which generated a fluorescent product, fluorescein. Using this technique, we obtained an intra-capillary precision with a coefficient of variation of 9.7%. In addition, the specificity study showed that the human IgG capillary immunosensor did not respond to rabbit IgG. Quantitative analysis was possible within the response range of 10 – 5000 ng mL⁻¹ anti-human IgG. This capillary immunosensor can act as a single analytical unit or can be integrated into a capillary array for multiple bioanalysis.

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