Anti-citrullinated protein antibody detection by hemagglutination

Published in ‘Rheumatology Advances in Practice’

20 January 2025, Authors: Ilmar Kruis, Jyoti Kumari, Annemarie van der Heijden, Amrah Weijn, Wilma Vree Egberts, Iris Rose Peeters, Noortje van Herwaarden, Martin Salden, Ger J.M. Pruijn

Abstract

Objectives: Anti-citrullinated protein antibodies (ACPA) play an important role in the classification of rheumatoid arthritis (RA) and can be detected by serological tests. Several ACPA-detection assays are available for clinical use, which are all based on ELISA(-like) assays with citrullinated peptides (CCP2) or alternative citrullinated proteins/peptides. We aimed to facilitate ACPA-detection in low-volume laboratories and resource-poor environments, we aimed to develop 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 psoriatic arthritis patients, and in healthy individuals.

Results: The agglutination mediator bound to erythrocytes, was reactive with ACPA, and induced hemagglutination 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 psoriatic arthritis (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 with only 3-21% of the PsA samples and with 1% of the healthy controls.

Conclusion: We conclude that the agglutination mediator allows the rapid and efficient detection of ACPA by hemagglutination in human blood samples and offers a low-threshold method for ACPA detection.

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Read the full article in: Rheumatology Advances in Practice

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