Multiple sclerosis (MS) is thought to be an autoimmune disease in which an unknown trigger initiates an immune response against brain proteins. This autoaggressive response causes the breakdown of the myelin sheaths that protect nerve axons, leading to impaired nerve conduction and subsequent neurodegeneration that are characteristic of MS. Many studies have attempted to determine the exact target within the brain. However, there appear to be multiple targets, which may change over time. No single study has examined all targets nor looked at how they can change over the course of the disease and whether these changes are related to the course of disease. We have approached this by using the single-cell resolution capability of the enzyme-linked immunospot assay to examine cytokine reactivity in MS patients in response to a very large set of overlapping peptides that span the two major proteins of myelin: myelin basic protein and proteolipid protein. Our goal was to use the enzyme-linked immunospot assay to perform comprehensive epitope mapping in relapsing-remitting MS patients in a longitudinal study to help define the role of myelin responses in disease progression.