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Journal of Biomedical Optics • Open Access

Confocal detection of planar homogeneous and heterogeneous immunosorbent assays
Author(s): Homanaz Ghafari; Yanzhou Zhou; Selman Ali; Quentin S. Hanley

Paper Abstract

Optically sectioned detection of fluorescence immunoassays using a confocal microscope enables the creation of both homo- and heterogeneous planar format assays. We report a set assays requiring optically sectioned detection using a model system and analysis procedures for separating signals of a surface layer from an overlying solution. A model sandwich assay with human immunoglobulin G as the target antigen is created on a glass substrate. The prepared surfaces are exposed to antigen and a FITC-labeled secondary antibody. The resulting preparations are either read directly to provide a homogeneous assay or after wash steps, giving a heterogeneous assay. The simplicity of the object shapes arising from the planar format makes the decomposition of analyte signals from the thin film bound to the surface and overlayer straightforward. Measured response functions of the thin film and overlayer fit well to the Cauchy-Lorentz and cumulative Cauchy-Lorentz functions, respectively, enabling the film and overlayer to be separated. Under the conditions used, the detection limits for the homogeneous and heterogeneous forms of the assay are 2.2 and 5.5 ng/ml, respectively. Planar format, confocally read fluorescence assays enable wash-free detection of antigens and should be applicable to a wide range of assays involving surface-bound species.

Paper Details

Date Published: 1 November 2009
PDF: 10 pages
J. Biomed. Opt. 14(6) 064022 doi: 10.1117/1.3268772
Published in: Journal of Biomedical Optics Volume 14, Issue 6
Show Author Affiliations
Homanaz Ghafari, Nottingham Trent Univ. (United Kingdom)
Yanzhou Zhou, Nottingham Trent Univ. (United Kingdom)
Selman Ali, Nottingham Trent Univ. (United Kingdom)
Quentin S. Hanley, Nottingham Trent Univ. (United Kingdom)

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