Single-step sandwich immunoreaction in a square glass capillary immobilizing capture and enzyme-linked antibodies for simplified enzyme-linked immunosorbent assay.

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.