Autoimmune diseases represent a complex and heterogeneous group of disorders characterized by persistent and pathological immune responses against self-components, leading to tissue damage and functional impairment. The core pathogenic mechanism lies in the breakdown of immune tolerance—the fundamental ability of the immune system to distinguish "self" from "non-self." Their pathogenesis results from the interplay between genetic susceptibility and environmental triggers. Among these diseases, autoantibodies serve not only as key pathogenic effector molecules but also as critical diagnostic and prognostic biomarkers.
Common autoimmune diseases classified according to anatomical site.[1]
Conventional therapeutic approaches for autoimmune diseases include broad-spectrum immunosuppressants (such as glucocorticoids and methotrexate) and antimalarial agents (e.g., hydroxychloroquine). In recent years, with a deeper understanding of the underlying pathogenic mechanisms, biologic agents and small-molecule targeted therapeutics have achieved revolutionary progress, enabling precise intervention at specific immunological checkpoints—including B-cell depletion, BAFF inhibition, co-stimulatory blockade, and suppression of multiple cytokine pathways (TNF, IL-6, IL-17/23, among others). The clinical pursuit of achieving durable remission, preserving organ function, and managing long-term complications through early recognition of preclinical states and precision-stratified treatment has become a tangible reality.[1]
1 Citrullination: A Unique Post-Translational Modification
Citrullination is a critical post-translational modification that plays a central role in the pathogenesis of autoimmune diseases, particularly rheumatoid arthritis (RA). This process is catalyzed by the peptidyl arginine deiminase (PAD) enzyme family, which converts arginine residues within proteins into citrulline. Although this chemical transformation does not alter the net charge of the amino acid, it significantly influences protein three-dimensional conformation, function, and intermolecular interactions.
Under physiological conditions, citrullination participates in various normal biological processes. However, under pathological states—especially within chronic inflammatory milieus—aberrant activation of PAD enzymes leads to excessive citrullination of numerous endogenous proteins. These modified "neoantigens" are recognized by the immune system, thereby breaching immune tolerance and eliciting the production of highly specific autoantibodies—known as anti-citrullinated protein antibodies (ACPA).
ACPA are among the most diagnostically valuable and prognostically significant biomarkers for rheumatoid arthritis. Their detection is crucial for the early diagnosis of RA, as they can be identified years before the onset of clinical joint symptoms, marking the preclinical phase of the disease.
2 Discovery and Design of Cyclic Citrullinated Peptides
The discovery and design of cyclic citrullinated peptides (CCP) have transformed the field of RA research, providing a key molecular tool for understanding its autoimmune pathogenesis and revolutionizing the diagnostic approach to the disease. However, the use of native citrullinated proteins as diagnostic antigens has long been hampered by numerous drawbacks, including complex sourcing, substantial batch-to-batch variability, and difficulties in standardization. Research revealed that the citrullinated sequences within natural filaggrin tend to adopt a stable β-hairpin conformation under physiological conditions. Recognizing that ACPA recognize a conformation-dependent epitope, investigators designed and synthesized cyclic citrullinated peptides by introducing covalent bonds (such as disulfide or amide bonds) between the N-terminus, C-terminus, or side chains of the peptide chain, thereby constraining the peptide into a rigid cyclic or hairpin-like conformation that mimics the native antigenic structure[2]. This conformational constraint significantly enhanced peptide stability and enabled more faithful representation of the antigenic epitopes present on in vivo citrullinated proteins. This design proved remarkably successful: anti-CCP assays demonstrate diagnostic performance far superior to that of rheumatoid factor (RF), achieving specificities exceeding 95% and sensitivities ranging from 65% to 80%, establishing them as a gold-standard diagnostic tool for RA[3].
3 Anti-CCP Antibodies
From a technological evolution perspective, anti-CCP antibody testing has undergone iterative optimization from first-generation to third-generation assays. Anti-cyclic citrullinated peptide antibodies (anti-CCP antibodies) represent one of the most specific and clinically valuable serological biomarkers in rheumatoid arthritis. They belong to the broader family of anti-citrullinated protein antibodies (ACPA), with their targets being artificially synthesized, conformationally stable cyclic citrullinated peptides that mimic the citrullinated epitopes formed on various endogenous proteins (such as fibrinogen, vimentin, and filaggrin) following PAD-mediated catalysis. Initial linear citrullinated peptides suffered from poor conformational flexibility and stability, resulting in inadequate detection sensitivity. The discovery of the β-hairpin structure and the subsequent synthesis of cyclic peptides directly gave rise to second- and third-generation CCP assay kits characterized by high sensitivity (65%–80%) and exceptional specificity (>95%), ultimately leading to their inclusion in the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria for RA[3].
4 From Peptide Antigens to Diagnostic Assays: The Application of CCP in In Vitro Diagnostics
The core value of CCP in in vitro diagnostics (IVD) lies in its function as a capture antigen for the detection of anti-cyclic citrullinated peptide antibodies (anti-CCP antibodies), providing a serological diagnostic tool for RA that combines high sensitivity with outstanding specificity. In modern clinical laboratories, CCP-based anti-CCP assays demonstrate remarkably robust performance: specificity typically exceeds 95%, far surpassing that of conventional rheumatoid factor (RF)—which can yield false-positive results in chronic infections, other connective tissue diseases, and even healthy elderly populations—while sensitivity reaches a clinically acceptable range of 65% to 80%[3].
With ongoing technological advances, the application formats of CCP in IVD continue to evolve. Traditional enzyme-linked immunosorbent assays (ELISA) typically employ only one or a limited number of CCP sequences, which may not comprehensively capture the highly heterogeneous ACPA repertoire present in RA patients. This multivalent antigen platform enables a more complete "profiling" of the ACPA reactivity signature in patient sera, holding promise for achieving more precise diagnosis, more refined disease subtyping, and more reliable prognostic prediction compared to single-CCP assays.