Foot-and-mouth disease virus (FMDV) causes an economically devastating disease of livestock that is controlled in endemic areas by vaccines containing intact inactivated FMDV particles. In this study, a novel monoclonal antibody named 5B6 has been identified and characterised, that permits the detection of all serotypes of FMDV via a conserved epitope near the N-terminus of the VP4 capsid protein. The antibody recognises intact virus particles known as 146S (the protective antigen) which contain VP4 and not dissociated capsids known as 12S (poorly protective antigen) which lack VP4. This allowed the development of a universal assay to specifically detect the protective antigen in vaccine samples using a simple ELISA. Such a test could be used to assess the quality of formulated vaccine following manufacture or prior to administration, or to assess unformulated vaccine antigen, and would be of great utility to enhance the effectiveness of FMD vaccination programmes.