Acute Rheumatic Fever (ARF) is a serious post-infectious sequela of a Group A Streptococcus (GAS) infection that can progress to rheumatic heart disease. ARF rates remain unacceptably high in Indigenous children in Australia and Māori and Pacific children in New Zealand, yet pathogenesis is poorly understood. During a GAS infection, immune dysregulation can result in the generation of immune cells and antibodies that bind to human proteins and contribute to ARF disease. The current dogma is that mimicry exists between GAS and human heart proteins providing a basis for cross-reactivity, but few studies have applied contemporary antibody profiling technologies to ARF. In this project, two high-throughput techniques were employed to identify novel human antigens that are reactive in ARF patient sera compared with matched healthy controls and GAS pharyngitis. These being Human Protein Arrays, containing between 9,000-16,000 full-length proteins and Phage Immuno-Precipitation Sequencing (PhIP-Seq), which displays the entire human proteome as 250,000 peptides 90 amino acids in length on the surface of phage molecules. From these two approaches, four autoantigens found in heart and connective tissue (PTPN2, DMD, a myosin associated protein and collagen 1) were selected for orthogonal validation by ELISA. For all four autoantigens, serum reactivity was significantly elevated in an expanded ARF cohort. Peptides corresponding to the specific epitopes of the myosin associated protein and collagen 1 detected by PhIP-Seq have also been synthesised for validating by ELISA. The microarray and ELISA techniques were restricted to measuring total IgG only, however Luminex bead-based assays allow for simultaneous detection of antibody isotypes and subclasses in a multi-plex format. To further explore the autoantibody features associated ARF, the autoantigens have been coupled to Luminex beads as both recombinant proteins, as well as long peptides, and this multiple autoantigen bead-based assay is undergoing optimisation.