Research paperMuscarinic type 3 receptor autoantibodies are associated with anti-SSA/Ro autoantibodies in Sjögren's syndrome

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- A muscarinic type 3 receptor (M3R)-GFP fusion protein over-expressed in a cell line enabled M3R autoantibody screening.
- The M3R-GFP In-Cell Western assay identified elevated anti-M3R in Sjögren's syndrome (SjS) patients compared to controls.
- Anti-M3R detection was associated with anti-SSA/Ro positivity in SjS patients.

Anti-muscarinic type 3 receptor autoantibodies (anti-M3R) are reported as potential inhibitors of saliva secretion in Sjögren's syndrome (SjS). However, despite extensive efforts to establish an anti-M3R detection method, there is no clinical test available for these autoantibodies. The purpose of this study was to propose inclusion of anti-M3R testing for SjS diagnosis through investigation of their prevalence using a modified In-Cell Western (ICW) assay. A stable cell line expressing human M3R tagged with GFP (M3R-GFP) was established to screen unadsorbed and adsorbed plasma from primary SjS (n = 24), rheumatoid arthritis (RA, n = 18), systemic lupus erythematosus (SLE, n = 18), and healthy controls (HC, n = 23). Anti-M3R abundance was determined by screening for the intensity of human IgG interacting with M3R-GFP cells by ICW assay, as detected by an anti-human IgG IRDye800-conjugated secondary antibody and normalized to GFP. Method comparisons and receiver-operating-characteristic (ROC)-curve analyses were performed to evaluate the diagnostic value of our current approaches. Furthermore, clinical parameters of SjS were also analyzed in association with anti-M3R. Anti-M3R was significantly elevated in SjS plasma in comparison with HC, SLE, or RA (P < 0.01). SjS anti-M3R intensities were greater than two-standard deviations above the HC mean for both unadsorbed (16/24, 66.67%) and adsorbed (18/24, 75%) plasma samples. Furthermore, anti-M3R was associated with anti-SjS-related-antigen A/Ro positivity (P = 0.0353). Linear associations for anti-M3R intensity indicated positive associations with focus score (R2 = 0.7186, P < 0.01) and negative associations with saliva flow rate (R2 = 0.3052, P < 0.05). Our study strongly supports our rationale to propose inclusion of anti-M3R for further testing as a non-invasive serological marker for SjS diagnosis.