Identification of Novel Autoantibodies in Patients with Ankylosing Spondylitis Using Human Protein Microarray

Matthew Presby¹, Mark J. Soloski¹, John A. Flynn², Clifton O. Bingham III¹, Michael M. Ward³ and Grant H. Louie⁴, ¹Johns Hopkins University, Baltimore, MD, ²Dept of Medicine, Johns Hopkins University, Baltimore, MD, ³Bldg 10 CRC Rm 4-1339, NIAMS/NIH, Bethesda, MD, ⁴Rheumatology, Johns Hopkins University, Baltimore, MD

Meeting: 2014 ACR/ARHP Annual Meeting

Keywords: Ankylosing spondylitis (AS), autoantibodies and autoantigens

Session Information

Title: Spondyloarthropathies and Psoriatic Arthritis - Pathogenesis, Etiology

Session Type: Abstract Submissions (ACR)

Background/Purpose: Ankylosing spondylitis (AS) is an immune-mediated disease for which the search for autoantibodies has been elusive. Conventional immunoserological approaches are slow and limited to the analysis of several dozen proteins at a time. In this pilot study, we used large-scale, high-throughput, protein microarrays to identify potential autoantibodies in AS.

Methods: Sera from patients who fulfilled the 1984 modified New York criteria for AS (N=20) were profiled using a human protein array composed of ~17,000 human proteins (CDI Laboratories, MD) and compared with healthy controls (N=18). Proteins were purified from Saccharomyces cerevisae, N-terminus tagged with GST-HisX6, spotted in duplicate on the array, imaged using a GenePix 4000B microarray scanner (Molecular Devices, CA), and analyzed using GenePix Pro software. Signal intensity, referred to as an A-score, was computed.

Results: AS patients were age (mean ± SD) 53.5 ± 12.1 years, 80% men, 100% HLA-B27 positive, with a disease duration (mean ± SD) of 28.6 ± 12.8 years. Healthy controls were age 55.7 ± 14.4 years and 50% men. Among 146 potential autoantigens detected in AS patients but not in healthy controls, we identified 6 with the highest frequency and signal intensity. These proteins were microtubule-associated serine/threonine-protein kinase 4 (MAST4), centriole, cilia and spindle-associated protein (CCSAP), SNF related kinase (SNFRK), calcium-binding protein 4 (CaBP4), protein MTG8 (MTG8), and eukaryotic elongation factor 2 kinase (EEF2K). The 3 most frequently targeted autoantigens were the serine/threonine kinases SNFRK, EEF2K, and MAST4. SNFRK and EEF2K were positive in 4/20 patients (20%). Three patients (15%) were positive for autoantibodies against MAST4. Two patients (10%) had antibodies to both MTG8 and CCSAP. The highest A-score observed was reactivity to CaBP4 by 1 patient (5%) (Figure 1). This was the lowest frequency autoantigen observed; however, CaBP4 reactivity was higher in the overall AS population compared to healthy controls. The images of each autoantigen were inspected and can be seen in Figure 2 for the patients with the five highest A-scores.

Conclusion: Using a high-throughput, protein microarray system, we have identified 6 novel autoantigens (SNFRK, EEF2K, MAST4, MTG8, CCSAP, and CaBP4) in AS patients, with an overall frequency of 5-20%. The detection of these AS-specific antibodies may allow for better understanding of disease pathogenesis and potential therapeutic targets. Further validation studies in larger numbers of patients are currently underway.

Disclosure:

M. Presby,
None;

M. J. Soloski,
None;

J. A. Flynn,
None;

C. O. Bingham III,
None;

M. M. Ward,
None;

G. H. Louie,
None.

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