A protein array platform for anti-citrulline antibodies in Rheumatoid Arthritis

Summary

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

Detail Information

Research Grants30

  1. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert 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. ..
  2. Intellectual and Development Disabilities Research Center
    Marc 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. ..
  3. Dissection of the ACPA response in African-Americans with Rheumatoid Arthritis
    S Louis Bridges; Fiscal Year: 2013
    ..These novel studies will provide important new information on the pathogenesis of RA in Af- Amer and may lead to innovative ways to diagnose, treat, or prevent this disease. ..
  4. JHU ICMIC PROGRAM
    Zaver 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. ..
  5. University of Maryland Greenebaum Cancer Center Support Grant
    Kevin J Cullen; Fiscal Year: 2013
    ..Reflecting our remarkable and continued growth, UMGCC seeks to renew its CCSG to enhance and expand its efforts in high-quality and clinically relevant cancer research. ..
  6. DIABETES AND ENDOCRINOLOGY RESEARCH CENTER
    Domenico Accili; Fiscal Year: 2013
    ....
  7. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  8. Southeast Regional Centers of Excellence for Biodefense &Emerging Infectious Di
    Philip Frederick Sparling; Fiscal Year: 2013
    ..SERCEB brings new investigators to the biodefense effort through a combination of educational programs, support of innovative new projects, and the synergistic interactions among its world-class investigators. ..
  9. Center for Gene Therapy of Cystic Firbosis
    John 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. ..
  10. Molecular Analyses and Interventions for Biodefense and Emerging Pathogens
    Olaf 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. ..
  11. BIOMARKERS OF EXPOSURE TO HAZARDOUS SUBSTANCES
    Bruce D Hammock; Fiscal Year: 2013
    ..abstract_text> ..
  12. UNMC EPPLEY CANCER CENTER SUPPORT GRANT
    Kenneth H Cowan; Fiscal Year: 2013
    ....