High Fidelity Cyclic Peptide Microarray to Diagnose Rheumatoid Arthritis
Authors: Krishnamurthy Hari , Jayaraman Vasanth , Krishna Karthik , Rajasekaran Karenah , Wang Tianhao , Bei Kang , Rajasekaran John , Elkon Keith 
Published: May 2021

Abstract
Introduction Antibodies reactive with cyclic citrullinated peptides (ACPA) have been included as a part of the ACR criteria for rheumatoid arthritis (RA) since 2010ii . However, due to the inability to capture the polydispersity of antibodies in RA without losing specificity, the ELISA assays cannot be used as definitive markers for diagnosis or prognosis. Current ELISA methods employ at most 30 unique cyclic citrullinated peptides for detection of ACPA. Apart from antibodies to citrullinated peptides, the serum from RA patients also contains antibodies to carbamylated antigens. Citrullinated and Carbamylated peptides hence show great promise for the improved diagnosis of RA. Here we tested whether a novel silicon-based combinatorial high-fidelity and high throughput cyclic peptide microarray that includes both citrullination and carbamylation modifications identifies RA with improved sensitivity compared to standard ELISA assays. Methods Using a cyclic modified peptide (CMP) library of more than 2 million sequences we tested 121 clinically diagnosed RA patients and compared binding profiles to corresponding linear epitopes and to commercially available CCP kits. Disease controls and samples from healthy individuals were also used to determine the specificity of the assay. The tests were performed using high throughput liquid handlers integrated with biochip imagers enabling high automation and a rapid turnaround time of 2.5 hrs. Since the citrulline and homocitrulline are very difficult to distinguish except by mass spectrometry, we will use the term Vibrant ACPA to include antibodies that react with either or both of these modified peptides in our assay. Results Using our CMP library, the sensitivity of RA detection was 95.04% with a specificity of 95.27% compared to linear peptides (Sensitivity 43.80% and Specificity 96.21%) and to commercial CCP kits (Sensitivity 66.94% and Specificity 89.20%). Inclusion of both citrullinated and carbamylated sequences provided increased sensitivity of testing (from 67% to 95%) as shown in Table 5. Some samples were only positive for carbamylated peptides and some only positive for citrullinated peptide indicating the importance of both peptide modifications.Conclusions These novel cyclic modified peptide (CMP) sequences have a high degree of accuracy in differentiating RA from controls, compared with standard serologic ELISA tests. Both citrullinated and carbamylated peptides are necessary for increasing the current sensitivity of RA testing. We also established here that a rigid conformation of the peptide is necessary for improved capture of antibodies by comparing cyclic and linear versions of the same peptide showing improved sensitivity with cyclisation. This high throughput pillar plate platform along with the 2 million data points generated per sample enable immune profiling on an unprecedented scale. While improved diagnostics is the primary outcome presented here, future identification of antigenic peptides that enable better prediction of prognosis and therapy would potentially improve outcomes in the affected population.