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Abstract Number: 0042

High-throughput Testing for Modified-protein Antibodies in Patients Diagnosed with “Seronegative” Rheumatoid Arthritis

Michael Richter1, Hari Krishnamurthy2, Sylvia Posso3, Jeffrey Carlin4 and Jane Buckner3, 1University of Washington, Mercer Island, WA, 2Vibrant Sciences, San Carlos, CA, 3Benaroya Research Institute at Virginia Mason, Seattle, WA, 4Virginia Mason Medical Center, Seattle, WA

Meeting: ACR Convergence 2021

Keywords: Anti-CCP, Anti-citrullinated Protein Autoantibodies (ACPAs), autoantigens, hla, rheumatoid arthritis

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Session Information

Date: Saturday, November 6, 2021

Session Title: RA – Etiology & Pathogenesis Poster (0011–0045)

Session Type: Poster Session A

Session Time: 8:30AM-10:30AM

Background/Purpose: Antibodies to citrullinated and other modified proteins play a critical role in the pathogenesis of rheumatoid arthritis (RA). The prevalence and degree of multi-site peptide reactivity of these antibodies in patients classified as “seronegative” is unclear.

Methods: An ultra-dense peptide array consisting of two million sequences was manufactured using photolithography. The peptide library was random but biased to include the sequences of filaggrin, fibrinogen, vimentin, collagen II, enolase, histones, and 14-3-3 eta with native, citrullinated, and carbamylated epitopes. A machine-learning algorithm identified 314 RA-specific peptides from a testing cohort of 140 RA patients, 480 healthy controls, and 136 disease controls based on signal-to-noise ratio. A validation cohort of 249 patients with RA (Table 1) was then analyzed on the basis of anti-cyclic citrullinated peptide (CCP) and rheumatoid factor (RF) positivity.

Results: The validation cohort included 106 patients (43%) who were negative for both CCP and RF (“seronegative”). Within this group, 25% had antibody binding to at least one site in the library of 314 peptides, compared with 98% of patients in the CCP and RF positive group (p< 0.0001). Only one control patient (1%) had any positive result. A heat-map of peptide reactivity is shown in Figure 1. Multi-site reactivity was more limited among the “seronegative” patients, with only 4% having antibody binding to at least 50 sites, compared to 80% in the CCP and RF positive group (p< 0.0001) (Figure 2). There was no significant difference in the ratio of antibodies to carbamylated vs. citrullinated proteins between “seronegative” and CCP positive patients. The presence of any HLA shared epitope allele was associated with positivity in this assay, with odds ratios of 3.56 (95%CI 1.99-6.37) for any binding and 4.22 (95%CI 2.22-6.37) for binding to greater than 50 sites.

Conclusion: This high-throughput assay demonstrated the presence of modified protein antibodies in a subset of “seronegative” patients, though multi-site reactivity was rare relative to CCP positive patients. This suggests that the mechanisms driving polyreactivity are features of seropositive RA and “seronegative” disease may have unique pathogenic features even when autoantibodies are present.

Table 1: Demographics of the validation cohort

Figure 1. Heat-map of peptide reactivity. Results determined through immunofluorescence with tyramide signal amplification. Columns represent individual patients with rows showing antibody binding to each peptide.

Figure 2. Number of peptide binding sites for every patient with at least one positive result. Each circle represents an individual patient.


Disclosures: M. Richter, None; H. Krishnamurthy, None; S. Posso, Janssen R&D, 5; J. Carlin, None; J. Buckner, Janssen R&D, 5.

To cite this abstract in AMA style:

Richter M, Krishnamurthy H, Posso S, Carlin J, Buckner J. High-throughput Testing for Modified-protein Antibodies in Patients Diagnosed with “Seronegative” Rheumatoid Arthritis [abstract]. Arthritis Rheumatol. 2021; 73 (suppl 9). https://acrabstracts.org/abstract/high-throughput-testing-for-modified-protein-antibodies-in-patients-diagnosed-with-seronegative-rheumatoid-arthritis/. Accessed January 27, 2023.
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