Antibodies against the M3 muscarinic acetylcholine receptor (M3R) are believed to have inhibitory effects on parasympathetic neurotransmission in patients with Sjögren's syndrome (SJS), leading to lacrimal and salivary glandular dysfunction. The second extracellular loop domain of M3R is regarded as the ligand binding site.
The aim of our study was to optimize an ELISA for the determination of antibodies against M3R synthetic peptide and to analyze sera from patients with SJS, patients with systemic lupus erythematosus (SLE) and healthy blood donors.
The M3R 25-mer peptide (KRTVPPGECFIQFLSEPTITFGTAI) was synthesized (Diagen, Ljubjana, Slovenia) and used as the antigen for anti-M3R ELISA. Synthetic peptide (10 mg/l) was used to coat Costar medium binding plates; the detection system was alkaline phosphatase/pNPP. Sera from 94 SJS patients (primary and secondary, age 17–77 years), 92 SLE patients (age 18–68 years) and 142 blood donors as controls (age 18–65 years) were tested.
According to the cutoff value, estimated at the 95 percentile of the controls (5/145 pos), positive values for anti-M3R were measured in 16/95 SJS and 8/92 SLE patients. Statistically significant differences in anti-M3R was found in patients with SJS compared with blood donors and compared with SLE patients (P = 0.0012 and P = 0.047, respectively). There was no significant difference between SLE patients and controls.
This was the first study of anti-M3R antibodies using the 25-mer synthetic peptide as the antigen on large, well defined groups of patients and blood donors, showing highly statistically significant elevation in antibodies in primary and secondary SJS compared with controls and SLE patients.