Sequential high-content profiling of the IgG-autoantibody repertoire reveals novel antigens in rheumatoid arthritis

Abstract

BackgroundThe aim was to identify novel diagnostic autoantibody candidates for rheumatoid arthritis (RA) by comprehensive screening for autoreactivity.MethodWe incubated 5892 recombinant proteins coupled to fluorescent beads, with patients’ sera for the detection of IgG-autoantibodies in three independent patient cohorts: A (n = 72 patients with established RA); B/B- (n = 116 patients with early RA (B) and n = 51 CCP-negative patients with early RA from B (B-)); and C (n = 184 patients with early seronegative RA), in comparison to matched healthy controls. Intersects of significantly increased autoantibodies as determined by the Mann-Whitney test were sought.ResultScreening of 5892 antigens in RA cohorts A and B, or the seronegative cohorts B- and C revealed intersects of 23 and 13 significantly increased autoantibodies, respectively. Reactivity to three antigens was increased in all cohorts tested: N-acetylglucosamine-1-phosphate transferase, gamma subunit (GNPTG), heterogeneous nuclear ribonucleoprotein A1-like 2 (HNRNPA1), and insulin-like growth factor binding protein 2 (IGFBP2).ConclusionsComprehensive sequential screening for autoantibodies reveals novel candidates for diagnostic markers in both seropositive and seronegative RA and suggests new fields of research into the pathogenesis of RA.

Profiling of IgG antibodies targeting unmodified and corresponding citrullinated autoantigens in a multicenter national cohort of early arthritis in Germany

“…A diagnosis of early "seronegative" RA, in this context defined as a lack of both RF and ACPA, may be particularly challenging. This "serological gap" [23] may progressively be closed by antibodies to post-translationally modified antigens such as antibodies to citrullinated or carbamylated proteins [9,10,24] and by the identification of hitherto unrecognized unmodified autoantigenic targets, as we and others have previously shown [12,13,18,25].…”

“…IgG reactivities against 390 unmodified recombinant proteins and 390 corresponding in vitro citrullinated recombinant proteins were assessed: Luminex bead-based antigen arrays were used for the multiplex analysis of IgG autoantibody reactivity as previously described [17,18]. In total, 390 antigens were selected based on literature data, and autoantibody reactivity data identified in previous high-content profiling studies in rheumatoid arthritis and SLE [18,19]. Additional proteins were chosen from multiple functional and disease pathways based on the gene ontology (GO) annotations within the Database for Annotation, Visualization, and Integrated Discovery (DAVID) version 6.8a [20].…”

Comprehensive Longitudinal Surveillance of the IgG Autoantibody Repertoire in Established Systemic Lupus Erythematosus

“…Bead‐based antigen arrays were used for the multiplex analysis of IgG autoantibody reactivity against 398 antigens representing 354 unique genes. In addition to well‐described SLE‐specific autoantigens, diagnostic antigens from other autoimmune diseases were selected based on literature data and autoantibody reactivity data identified in previous high‐content profiling studies in rheumatoid arthritis and SLE . The full list of antigens is provided in Supplementary Table 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40788/abstract.…”

“…For some established SLE antigenic complexes, multiple clones were available (U1 RNP, n = 6; Sm, n = 10; ribosomal P, n = 5), representing different antigens within the target protein complex (Supplementary Table 1). Protocols for the expression, purification, and bead‐coupling of proteins were applied as previously described . Briefly, antigens were produced in Escherichia coli , purified, and covalently coupled with magnetic carboxylated color‐coded beads (MagPlex microspheres; Luminex).…”

Proteomic analysis to define predictors of treatment response to adalimumab or methotrexate in rheumatoid arthritis patients

“…The full list of antigens is provided in the Supplementary Information. Detailed methodology for protein expression and multiplex autoantibody measurement has previously been described [15]. In brief, antigens were produced in E. coli, purified and covalently coupled to magnetic carboxylated colourcoded beads (MagPlex TM microspheres, Luminex Corporation, Austin, Texas).…”