A protein array platform for anti-citrulline antibodies in Rheumatoid Arthritis
Principal Investigator: Ji Qiu
Abstract: DESCRIPTION (provided by applicant): Anti-citrulline antibodies are specific and predictive of rheumatoid arthritis (RA). Seropositivity against citrulline is clinically assayed using cyclic citrullinated peptide (CCP) ELISA. Despite being an excellent proxy assay for diagnosis, anti-CCP positivity does not reveal any information about the actual underlying antigens that elicited the immune response. Recent studies demonstrated the value of identifying autoantibodies to particular antigens in the elucidation of RA etiology. Furthermore, the identification of specific citrullinated antigens might help improve the assay performance of anti-CCP test. Unfortunately, only a few citrullinated antigens have been discovered in the past several decades. Developing a platform that will identify antibodies against citrullinated proteins will not only help understad the disease pathogenesis but also improve diagnosis. Traditional protein immuno-chemistry methods to identify citrullinated antigens suffer drawbacks such as low throughput, poor reproducibility, inadequate quantification and low resolution. Commercial protein arrays are expensive, lack equal representation of candidate antigens and are not compatible with post-translational modifications such as citrullination, which requires harsh conditions. We propose a cover capture protein microarray platform where proteins are expressed in silicon microwells and captured as naked protein by ligands on the cover slides. Our development will be based on our innovative NAPPA protein microarray platform to circumvent challenges associated with commercial protein arrays for spotting purified recombinant proteins. NAPPA involves printing full length cDNAs corresponding to proteins of interest on the microarray substrate and then transcription/translation in situ at the time of assay. Cover capture NAPPA will enable just-in-time proteins expression and citrullination on array post-translationally without the interference of NAPPA printing mixture components. Captured proteins will be presented on standard microscopic slides and the employment of high affinity tag/ligand will enable a variety of post-translational modifications on array including citrullination. Sero- reactivity against multiple citrullinated proteins can then be assessed in parallel. We will establish this platform to profile anti-citrullinated protein antibodies in RA at the proteome level and to map immuno-dominant epitopes of citrullinated autoantigens on array with the key advantages being high-throughput, multiplexed, quantitative, and low-cost so that enough samples can be assayed to draw statistical sound conclusion. Our goal is to develop a high-throughput platform to discover additional antigens, when citrullinated, can be recognized by antibodies in RA patients at the proteome level. We believe cover-capture NAPPA will enable this discovery and greatly benefit RA research to understand disease pathogenesis and develop more sensitive diagnostics enabling patient stratification based on autoantigens. Furthermore, what we propose to develop can be applied to studies in understanding the biology of other post-translational modifications.
Funding Period: 2012-09-24 - 2014-08-31
more information: NIH RePORT
- CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASERobert S Sandler; Fiscal Year: 2013..Through all of its activities, the Center improves communication, promotes collaboration, develops careers and generally enriches the intellectual climate for digestive disease research. ..
- Intellectual and Development Disabilities Research CenterMarc Yudkoff; Fiscal Year: 2013..5 million from NICHD). The Center includes an excess of 70 Penn faculty at 15 departments at the Schools of Medicine, Veterinary Medicine, Nursing, the Wistar Institute, and the College of Arts and Sciences. ..
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- JHU ICMIC PROGRAMZaver M Bhujwalla; Fiscal Year: 2013..The Career Development Component is structured with the purpose of creating independently funded investigators who will, in the future, become leaders in the field. ..
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- Center for Gene Therapy of Cystic FirbosisJohn F Engelhardt; Fiscal Year: 2013..These efforts have led to numerous basic and applied research findings that have enhanced the utility of gene therapies to both study and treat genetic diseases. ..
- Molecular Analyses and Interventions for Biodefense and Emerging PathogensOlaf Schneewind; Fiscal Year: 2013..Research and training at the GLRCE is governed by a mechanism involving ongoing review of scientific excellence and translational goals, inter-institutional advisory boards and external scientific advisory bodies. ..
- BIOMARKERS OF EXPOSURE TO HAZARDOUS SUBSTANCESBruce D Hammock; Fiscal Year: 2013..abstract_text> ..
- UNMC EPPLEY CANCER CENTER SUPPORT GRANTKenneth H Cowan; Fiscal Year: 2013....