# Insights into the detection of AMPA cross-reactivity: comparing cyclic peptide- to protein-based assays

## TL;DR

This study compares how different assay types detect cross-reactivity of autoantibodies in rheumatoid arthritis patients, showing that cyclic peptides reveal more cross-reactivity than protein-based methods.

## Contribution

The study reveals how antigen backbone variations affect the detection of AMPA cross-reactivity in rheumatoid arthritis.

## Key findings

- CXP2-based assays show higher cross-reactivity to multiple PTM residues compared to FCS-based assays.
- Modified FCS captures AMPAs with less cross-reactive epitope recognition than modified peptides.
- 61.2% of samples reacted to both citrullinated and carbamylated residues on CXP2, but only 54.0% on FCS.

## Abstract

Autoantibodies targeting antigens carrying distinct post-translational modifications (PTMs), including citrullinated, carbamylated, and acetylated residues, are characteristic for rheumatoid arthritis (RA). 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.

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.

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.

Antigen backbones influence the detection of AMPA cross-reactivity. Gaining a better understanding of how PTM backbones affect this detection could provide insights into the structural basis of AMPA reactivity, and refine data interpretation by highlighting how assay choice influences results.

The online version contains supplementary material available at 10.1186/s13075-025-03591-y.