When inflammation occurs in the body, some proteins are altered in a process called citrullination and these altered forms can prompt an immune response from the body, where antibodies attack the body causing autoimmune diseases like rheumatoid arthritis (RA).

For that reason, tests that spot antibodies to citrullinated proteins are already used to diagnose the disease and while tests for individual proteins usually have a relatively low diagnostic sensitivity, a more general test called cyclic-citrullinated peptide (CCP) detects synthetic citrullinated peptides, identifies many more RA cases.

Scientists at the Kennedy Institute of Rheumatology (Oxford, UK) screened a cohort comprised of 20 British patients with RA and 20 healthy individuals. The 101 pre-RA cases and 326 matched controls were identified in a nested case-control study in four Southern European cohorts. One thousand nine hundred and eighty-five cases of RA and 160 controls were from the Swedish population-based case-control. Two hundred and eighty-seven patients with RA and 330 control patients with osteoarthritis (OA) were from the USA.

Peptides were analyzed on a liquid chromatography–tandem mass spectrometry (LC-MS/MS) workflow comprising a Dionex Ultimate 3000 nLC system coupled to a Q-Exactive mass spectrometer (Thermo Scientific; Waltham, MA, USA). Differential Scanning Fluorimetry and recorded on the real-time polymerase chain reaction ViiA7system (Applied BioSytems; Foster City, CA, USA) and circular dichroism was performed and recorded on a J-815 CD spectrophotometer (Jasco; Easton, MD, USA). Antibodies to cyclic peptides containing citrullinated sites were screened in sera from patients with RA by enzyme-linked immunosorbent assay (ELISA).

Two immunodominant peptides citrullinated tenascin-C 1 (cTNC1, VFLRRKNG-cit-ENFYQNW) and cTNC5 (EHSIQFAEMKL-cit-PSNF-cit-NLEG-cit-cit-KR) were identified. Antibodies to both showed limited cross-reactivity with anticitrullinated protein antibody (ACPA) epitopes from α-enolase, vimentin and fibrinogen, and no reactivity with citrullinated fibrinogen peptides sharing sequence homology with fibrinogen-like globe (FBG). cTNC5 antibodies were detected in 18% of pre-RA sera, and in 47% of 1,985 Swedish patients with RA and 51% of 287 North American patients with RA. The specificity was 98% compared with 160 healthy controls and 330 patients with osteoarthritis.

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Kennedy Institute of Rheumatology
Thermo Scientific
Jasco