An Opsono-Adherence Assay for Assessing the Opsonization of Encapsulated Bacteria by Anti-Capsule Antibodies
Trascrizione
Take a suspension of chemically-killed, fluorescently labeled Bacillus anthracis.
The bacterial capsule, mainly composed of poly-gamma-glutamic acid, provides protection against phagocytosis.
Take non-human primate sera, heat-inactivated to destroy complement proteins. These sera, isolated from animals immunized with a bacterial-capsule-based vaccine, contain anti-capsule antibodies.
Add the chemically killed bacterial suspension to the sera. The antibodies specifically bind to the bacterial capsules, facilitating bacterial opsonization and marking them for elimination by phagocytic cells. Add standard complement proteins to enhance opsonization.
Transfer the opsonized bacteria to adherent macrophages — phagocytic cells. Incubate briefly.
The antibodies and complement proteins on the bacteria bind to macrophage Fc receptors and complement receptors, respectively, promoting bacterial adherence.
Remove media. Wash with buffer to remove non-adhered bacteria.
Fix the cells to preserve cellular integrity.
Use a fluorescent stain to label the macrophages.
Using a fluorescence microscope, observe fluorescent bacteria adhered to differently-fluorescent macrophages, confirming effective bacterial opsonization by anti-capsule antibodies.
