Detection of Sparse Botulinum Toxin A Binding Sites Using Fluorescent Latex Microspheres

Abstract

The most potent toxins known are produced by strains of Clostridium botulinum. To paralyze the vertebrate neuromuscular junction, the toxins bind selectively to nerve endings, translocate into the presynaptic terminal, and hydrolyze proteins of the exocytotic apparatus, thus inhibiting the release of acetylcholine. Our goal was to develop a convenient, reliable technique to detect specific binding of botulinum toxin A to its targets, a technique that could be easily modified to detect the binding sites of other ligands as well. Our method utilized fluorescent latex microspheres and is theoretically capable of detecting a single binding site at the light microscopic level.Non specific binding sites on 7-μm thick sections of unfixed, cryosectioned mouse diaphragm were first blocked with 20% goat serum in phosphate-buffered saline (GS/ PBS). We incubated the diaphragm for 1 hr at 22° with various concentrations of botulinum toxin A in GS/PBS, followed by incubation with rabbit anti-botulinum toxin A antiserum, biotin-labeled goat anti-rabbit antibody, and finally avidin-labeled, 0.03-μm diameter, fluorescent latex microspheres.As expected, binding was localized to the area of the neuromuscular junction. Binding was also observed in association with axons innervating some junctions. We could detect binding on diaphragms that were exposed to as little as 10 pM botulinum toxin A, which is in the low range of effective in vitro doses that block neuromuscular transmission. This is a convenient, sensitive, and specific technique for detecting botulinum toxin A binding sites that is easily modifiable for the detection of binding sites of other ligands as well. (The J Histotechnol 22:113, 1999)