Discovery of Two Novel Immunoepitopes and Development of Peptide-based Sarcoidosis Immunoassay

Rationale: Sarcoidosis is a systemic granulomatous disorder associated with hypergammaglobulinemia and the presence of autoantibodies. The specific antigens initiating granulomatous inflammation in sarcoidosis are unknown and there is no specific test available to diagnose sarcoidosis. To discover novel sarcoidosis antigens, we developed a high-throughput T7 phage display library derived from the sarcoidosis cDNA and identified numerous clones differentiating sarcoidosis from other respiratory diseases. After clone sequencing and homology search, we identified two epitopes (Cofilinμ and Chain A) that specifically bind to serum IgGs of sarcoidosis patients. Objectives: To develop and validate an epitope-specific IgG-based immunoassay specific for sarcoidosis. Methods: We chemically synthesized both immunoepitopes (Cofilinμ and Chain A), and generated rabbit polyclonal antibodies against both neoantigens. After extensive standardization, we developed a direct peptide ELISA and measured epitope-specific IgG in sera of 386 subjects including, healthy controls (n=100), three sarcoidosis cohorts (n=186), pulmonary tuberculosis (n=70) and lung cancer (n=30). Measurements and Main Results: To develop a model to classify sarcoidosis from other groups, data were analyzed using five-fold cross-validation when adjusting for confounders. The Cofilinμ IgGs model yielded a mean sensitivity, specificity, and positive and negative predictive value (PPV, NPV) of 0.97, 0.9, 0.9 and 0.96, respectively. Those same measures for Chain A IgG antibody were 0.9, 0.83, 0.84 and 0.9 respectively. Combining both biomarkers improved AUC, sensitivity, specificity, PPV and NPV. Conclusions: These results provide a novel immunoassay for sarcoidosis. The discovery of two neoantigens facilitates the development of biospecific drug discovery and the sarcoidosis-specific model.