Abstract
Nano- and microparticles have long been used for the delivery of drugs and are currently being evaluated as vaccine delivery systems. Particulates can elicit potent immune responses, either by direct immuno-stimulation of antigen presenting cells (APC) or/and by delivering antigen to specific cellular compartments and promoting antigen uptake by appropriate stimulatory cell types. Herein, we describe a detailed method for the preparation of a novel nanoparticle-based antigen delivery system which induces strong cellular and humoral immune responses in mice and sheep. This simple system is based on the use of 40 nanometer (nm) inert solid carrier beads to which antigen is covalently coupled before injection. Covalent conjugation of antigen to the nanobeads, assessment of conjugation efficiency, characterisation and measurement of in vivo immunogenicity by cytokine ELISPOT (to measure antigen-specific T-cell responses) and ELISA (to measure antibody titers), are described. Emphasis is placed on providing trouble-shooting advice to enable the reproducible production of soluble nano-size formulations that do not suffer from common problems such as aggregation, as well as understanding the causes and thus avoiding a range of prevalent technical problems that occur when using immune response detection assays, such as the cytokine ELISPOT assay and ELISA.
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