Development of multiplex Cell-Based Assays for detection of autoantibodies and biomarkers in rheumatic autoimmune diseases: Systemic Sclerosis and Immune-Mediated Necrotizing Myopathy

• Grant number: 23/17946-6
• Support Opportunities: Regular Research Grants
• Start date: November 01, 2024
• End date: October 31, 2027
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Cristiane Kayser
• Grantee: Cristiane Kayser
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated researchers: Gerson Dierley Keppeke

Abstract

Introduction: Systemic sclerosis (SSc) and systemic autoimmune myopathies (SAMs) are rare rheumatic autoimmune diseases whose diagnosis might be a challenge. The presence of serum autoantibodies (AAbs) is hallmark of the immune abnormalities of rheumatic diseases and have an important role in SSc and SAMs diagnosis. In SSc, about 10 most common AAbs are found, and >95% of the SSc patients present at least one in the serum. Moreover, in SSc, the presence of specific AAbs correlates with the clinic subtype and with the disease prognosis. Among patients with SAMs, about 20 to 30% are classified as Immune-Mediated Necrotizing Myopathy (IMNM). Two autoantibodies (AAbs) are considered specific biomarkers of IMNM: anti-HMGCR and anti-SRP. These AAbs seems to contribute to the disease pathogenesis, thus the detection of those in the patient in essential for the diagnosis of IMNM. Recently, a new Cell-Based Assay (CBA) for detection of the SSc related AAb against fibrillarin was developed, with an innovative strategy. The fibrillarin gene was fused to a transmembrane signal (TMS), and then transgenic expressed in HEp-2 cells, it relocates to cell membrane in a receptor-like fashion. The cell was then used as substrate in indirect immunofluorescence assay (IFA). Staining of the cells expressing TMS-fibrillarin indicate the presence of the anti-fibrillarin AAb in the respective sample. Objectives: In this study, we aim to develop a CBA multiplex for the detection of AAbs associated to SSc, including RNAP-III, Topo-I, Ku, Nor-90, PM-Scl-75/100, Fibrillarin and Th/To and the Aabs HMGCR and SRP associated with IMNMMaterial and methods: We will fuse the TMS to SSc-related autoantigens (RNAP-III, Topo-I, Ku, Nor-90, PM-Scl-75/100, Fibrillarin and Th/To) and with HMGCR and SRP to generate a multiplex CBA to detect the respective AAbs in samples from IMNM and SSc. For validation of the assays, 100 samples from SAMs patients and 150 samples from SSc will be collected and analyzed in the respective multiplexed CBAs. To evaluate the performance of the new test, they will be compared to available commercial assays. In addition, the results will be compared to demographic and clinical variable of the patients.Expected results: We hope to develop assays with improved sensibility, lower costs and easy execution protocols, when compared with commercial solid-phased assays currently applied to detect these AAbs. (AU)