Background: In rheumatoid arthritis (RA), anti-citrullinated protein/peptide antibodies (ACPAs) are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR) analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement activating peptide were used to induce selective depletion of ACPA producing B cells. Results: KD values of affinity purified ACPA IgGs varies between 10-6-10-8 M and inversely correlate with disease activity. Based on their cross-reaction with citrulline-peptides we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two-two copies, separated with a short, neutral spacer. This peptide detects antibodies in RA sera with 66 % sensitivity and 98 % specificity in ELISA and is recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targets and depletes ACPA producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed on the basis of ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.