Applications of Confocal Microscopy and Scanning Electron Microscopy to the Localization of Immunoreagents Used in Medical Diagnostic Systems

Abstract

Medical diagnostic assays often are based on the immobilization of immunolabeling reagents on solid substrates such as polystyrene beads, microparticles, or membranes. The distribution of immunoreagents on or within these substrates has a significant effect on product performance. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) can be used to localize immunological reagents on beads and other surfaces. In this paper we describe examples in which CLSM and SEM were used to assist in the design and troubleshooting of three diagnostic systems.A protective overcoat is used on HIV antibody-coated 0.25“ polystyrene beads in an HIV screening assay. To visualize topographical detail of the antibody distribution on beads with and without the overcoat, beads were labeled with a fluorescent secondary antibody and examined by CLSM. On overcoated beads, the distribution of HIV antibody was relatively uniform; but on non-overcoated beads, HIV antibody distribution was patchy and located mostly in low areas of the bead surface (Fig. 1), evidence that the overcoat provided protection for the HIV antibody. In the same assay system, performance was enhanced by using beads coated with a mixture of HIV antibodies from two IgG subclasses. The relative distribution of the two primary antibodies on the bead surface was demonstrated using isotype-specific secondary antibodies and CLSM.