microRNA- and Autoantibody-based Multiplex Assay for Systemic Lupus Erythematosus

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 DESCRIPTION (provided by applicant): Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with worldwide distribution. Despite medical treatment, morbidity and mortality from renal disease are still common in lupus patients. However, early diagnosis and prompt treatment can significantly improve long-term prognosis. Anti-double stranded (ds) DNA autoantibodies are a serologic hallmark of patients with SLE. In addition, circulating microRNAs (miRNAs) have been shown recently to be systematically altered, unveiling miRNA signatures with diagnostic utility. Growing evidence suggests that a multi-marker strategy, containing a combination of biomarkers with high clinical sensitivity and specificity, may enhance diagnostic and prognostic accuracy in the future compared to single marker tests. To support efforts of identifying the most informative biomarker panels, reliable next- generation platform technologies are needed that permit multiplexed detection of both protein and nucleic acid targets in small samples and are suitable for automation and integration into the clinical laboratory work flow. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for an ultrasensitive and - specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with high single-well multiplexing potential, minimal sample requirements, and simplified handling procedures (no separation/washing and amplification steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby target recognition molecules are tagged with different color fluorescent dyes (and quenchers). NTI recently achieved extension from 2-color to 4-color ALEX, substantially expanding its multiplexing power, and demonstrated diagnostic utility for direct protein as well as miRNA quantification. Furthermore, recent work by Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, termed "single molecule optofluidics", and ii) enhanced throughput using a multi-foci excitation/detection geometry. NTI's long-term goal is to develop rapid, highly multiplexed, ultrasensitive and -specific, as well as fully automated, nucleic acid- and protein-based diagnostic tests that require minimal sample sizes. Here, we propose assay development and clinical validation of a next-generation test with significantly improved diagnostic, prognostic, and treatment- guiding properties, implementing a panel of autoantibody and miRNA biomarkers, and overcoming limitations of current SLE testing. Our Specific Aims are: 1. Initial reagent development for a multiplex miRNA & autoantibody-based next-generation test for SLE 2. Separate as well as multiplexed biomarker detection and quantification using spiked samples 3. ALEX-based analysis of 42 archived clinical samples and cross-validation to ELISA and qPCR methods SBIR Phase II will propose assay expansion to include more markers, miniaturization, and development of a user-friendly, "sample in - answer out" diagnostic system offering significant cost and patient sample savings.