Use of receptors immobilized on microspheres to identify ligand binding sites in tissue sections: detection of lymph node ligands for L-Selectin

Abstract

Particulate microspheres bearing immobilized probes can be used to identify ligands expressed by cells and require only brightfield microscopy for detection. There are distinct advantages to using microspheres to detect low affinity interactions; microspheres require no secondary amplification or detection procedures subsequent to the binding interaction, reducing opportunities for detachment of bound probe, and concentrating probes on microspheres may greatly increase binding avidity. Selectin leukocyte-endothelial adhesion molecules undergo low affinity binding to ligands, and these interactions may be difficult to detect with standard techniques. The aim of this study was to determine if immobilizing recombinant L-Selectin on microspheres would facilitate detection of specific tissue ligands. Microspheres were incubated with sections of rabbit peripheral lymph node in a modified Stamper-Woodruff assay, and binding was assessed by brightfield microscopy. L-Selectin-IgG microspheres bound to high endothelial venules, known to be sites of expression for L-Selectin ligands. Specificity was indicated by the lack of binding of microspheres coated with control protein, and inhibition of binding by antibody to L-Selectin and by competitive antagonists of L-Selectin ligand interactions.