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These anti-modified protein antibodies (AMPAs) are typically detected using enzyme-linked immunosorbent assays (ELISAs), with peptides or protein antigens carrying these modifications. AMPAs exhibit significant cross-reactivity towards multiple PTMs, and increased cross-reactivity before disease onset may serve as a biomarker of disease progression. However, the impact of antigen backbone variations on cross-reactivity detection remains unclear. Therefore, we investigated how PTM-backbone variations affect AMPA-reactivity detection.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>Sera of 608 RA patients from the Early Arthritis Clinic (EAC) were measured for AMPA reactivity using modified fetal calf serum (FCS)- and cyclic peptide (CXP2)-based ELISAs. To investigate cross-reactivity patterns, we isolated AMPAs from serum using either modified FCS or peptides and assessed the reactivity of the isolated antibodies towards three different PTMs.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>CXP2-based assays reveal a higher proportion of patients with serum reactivity against multiple PTM residues, while FCS-based assays exhibit a more restricted serological profile. When comparing responses to citrullinated versus carbamylated backbones, 61.2% of samples reacted to both PTM-residues on CXP2, while on FCS this percentage significantly decreased to 54.0%. The antigen backbone also influences AMPA isolation, as modified FCS captures AMPAs with a more restricted, less cross-reactive epitope recognition profile compared to those captured with modified peptides.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>Antigen backbones influence the detection of AMPA cross-reactivity. Gaining a better understanding of how PTM\u00a0backbones affect this detection could provide insights into the structural basis of AMPA reactivity, and refine data interpretation by highlighting how assay choice influences results.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/s13075-025-03591-y","type":"journal-article","created":{"date-parts":[[2025,7,7]],"date-time":"2025-07-07T06:01:17Z","timestamp":1751868077000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Insights into the detection of AMPA cross-reactivity: comparing cyclic peptide- to protein-based assays"],"prefix":"10.1186","volume":"27","author":[{"given":"Roxane","family":"Biersteker","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aegli","family":"Athanasiadou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stef","family":"van der Meulen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tineke J.","family":"van Wesemael","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linda M.","family":"Slot","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Theresa","family":"Kissel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ren\u00e9 E. 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All participants provided written informed consent.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"ChatGTP-4o was used exclusively to improve readability and refine the language in this document. All scientific content was thoroughly reviewed and validated by all authors following each edit, ensuring that the accuracy and integrity of the research remained intact.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declaration of generative AI and AI-assisted technologies in the writing process"}},{"value":"Not applicable.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"L.A.T. and R.E.M.T. are mentioned as inventors on a patent on ACPA IgG V-domain glycosylation. The other authors declare no conflict of interest.","order":5,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"138"}}