Anti-citrullinated protein antibody detection by hemagglutination.

Objectives: ACPAs play an important role in the classification of RA and can be detected by serological tests. Several ACPA detection assays are available for clinical use, which are all based on ELISA(-like) tests with citrullinated peptides (CCP2) or alternative citrullinated proteins/peptides. We aimed to facilitate ACPA detection in low-volume laboratories and resource-poor environments by developing a rapid and easy-to-perform test. Methods: We designed and generated an agglutination mediator by protein engineering. A single-chain antibody fragment that binds to glycophorin A, one of the major surface proteins of erythrocytes, was conjugated to a CCP2-like peptide. This agglutination mediator was used to assess ACPA in RA and PsA patients and in healthy individuals. Results: The agglutination mediator bound to erythrocytes was reactive with ACPA and induced haemagglutination in an ACPA-dependent fashion, which can be detected by the naked eye. The applicability was assessed by the analysis of fresh blood samples from 204 RA patients, 77 PsA patients and 100 healthy individuals. Agglutination was observed in up to 61% of the RA samples, which correlated well with the results obtained with a standardized anti-CCP2 ELISA (63-67%). Depending on the minimal agglutination score, agglutination was observed in only 3-21% of the PsA samples and in 1% of the healthy controls. Conclusion: We conclude that the agglutination mediator allows rapid and efficient detection of ACPA by haemagglutination in human blood samples and offers a low-threshold method for ACPA detection.