Affinity Purification and Comparative Biosensor

18 Background: In rheumatoid arthritis (RA), anti-citrullinated protein/peptide antibodies (ACPAs) 19 are responsible for disease onset and progression, however, our knowledge is limited on ligand 20 binding affinities of autoantibodies with different citrulline-peptide specificity. 21 Methods: Citrulline-peptide specific ACPA IgGs were affinity purified and tested by ELISA. 22 Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon 23 resonance (SPR) analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide 24 and a complement activating peptide were used to induce selective depletion of ACPA producing 25 B cells. 26 Results: KD values of affinity purified ACPA IgGs varies between 10-6-10-8 M and inversely correlate 27 with disease activity. Based on their cross-reaction with citrulline-peptides we designed a novel 28 multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two-two copies, separated with a 29 short, neutral spacer. This peptide detects antibodies in RA sera with 66 % sensitivity and 98 % 30 specificity in ELISA and is recognized by 90% of RA sera, while none of the healthy samples in SPR. 31 When coupled to nanoparticles, the multi-epitope peptide specifically targets and depletes ACPA 32 producing B cells ex vivo. 33 Conclusions: The unique multi-epitope peptide designed on the basis of ACPA cross-reactivity 34 might be suitable to develop better diagnostics and novel therapies for RA. 35