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.
Graduate School of Engineering, Osaka Prefecture University, Japan.
Erina
E
Tsutsumi
Terence G
TG
Henares
Shun-ichi
S
Funano
Kunio
K
Kawamura
Tatsuro
T
Endo
Hideaki
H
Hisamoto
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D005898:Glass / Q000737:chemistry; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry
D000888:Antibodies, Anti-Idiotypic; D000936:Antigen-Antibody Complex; C048844:anti-IgG; D011092:Polyethylene Glycols; D000469:Alkaline Phosphatase