||Acute Rheumatic Fever (ARF) is an autoimmune disease occurring in individuals following untreated group A streptococcal infection believed to be triggered by antibodies to bacterial components that cross-react with human tissues. A multiplexed immunoassay was developed for the simultaneous quantitation of antibodies to nine different streptococcal related antigens including streptolysin O (SLO), DNase B, Collagens I and IV, fibronectin, myosin, group A carbohydrate, M6 protein, and C5 peptidase. Development of the assay involved testing of several different types of microspheres as well serum diluent formulations in an effort to reduce nonspecific binding of antibodies. Optimal coupling conditions for both protein and carbohydrate antigens to the microspheres were also determined. Monoclonal antibodies were used to determine antigen coupling efficiencies and cross-reactive epitopes of the different antigens. The multiplexed assay was then used to quantitate antibody responses in 49 ARF, 58 pharyngitis patients, and age and sex matched controls in samples collected at initial disease onset, and at 4 weeks, 6 months, and 1 year after diagnosis. Through the selection of microspheres and serum diluent, nonspecific antibody binding was reduced by 17%. Different formulations of the coupling buffer were found to greatly influence the efficiency of coupling antigens to the carboxylated microspheres. Monoclonal antibodies against the different antigens demonstrated assay specificity as well as sensitivities of less than 1 ng/ml of antibody. Antibody responses were significantly higher for SLO, DNase B, M6 protein, group A carbohydrate, and the cross-reactive antigens collagen I and myosin in ARF compared to pharyngitis patients (p<0.05). Moreover, we found significantly elevated antibody responses in the ARF patients with rheumatic heart disease (RHD) to fibronectin and collagen I, compared to ARF patients without heart disease. The major differences between the ARF patients with and without carditis appear to be in the immune response to the putative heart valve components, collagen I and fibronectin.