The natural host for Ebola virus, presumed to be an animal, has not yet been identified despite an extensive search following several major outbreaks in Africa. A straightforward approach used to determine animal contact with Ebola virus is by assessing the presence of specific antibodies in serum. This approach however has been made very difficult by the absence of specific reagents required for the detection of antibodies from the majority of wild animal species. In this study, we isolated a human monoclonal antibody Fab fragment, KZ51, that reacts with an immunodominant epitope on Ebola virus nucleoprotein (NP) that is conserved on all four Ebola virus subtypes. The antibody KZ51 represents a major specificity as sera from all convalescent patients tested (10/10) and sera from guinea pigs infected with each of the four Ebola virus subtypes competed strongly with KZ51 for binding to radiation-inactivated Ebola virus. These features allowed us to develop a novel assay for the detection of seroconversion irrespective of Ebola virus subtype or animal species. In this assay, the binding of KZ51 Fab-phage particles is used as an indicator assay and the presence of specific antibodies against Ebola virus in sera is indicated by binding competition. A prominent feature of the assay is that the Fab-phage particles may be prestained with a dye so that detection of binding can be directly determined by visual inspection. The assay is designed to be both simple and economical to enable its use in the field.