Biosensor analysis of anti citrullinated protein/peptide antibodies (ACPA) affinity

Anti-citrullinated protein/peptide antibodies (ACPA) are detected in rheumatoid arthritis (RA) sera and because of their strict association with the disease are considered marker antibodies, probably endowed with pathogenic potential. Antibody affinity is one of the parameters affecting pathogenicity. Three diagnostic citrullinated peptides (VCP1 and VCP2, derived from EBV-encoded proteins; HCP1 derived from histone H4) were synthesized as tetrameric Multiple Antigen Peptides and immobilized on sensor chips CM5-type in a Biacore® T100. Specific binding of purified antibodies from RA patients to the three peptides was analyzed by surface plasmon resonance using as controls two arginine containing sequences. Employing a 1:1 binding model for affinity constants calculation, ACPA interacted with VCP1 and VCP2 with lower apparent affinity (10-6 M > KD > 10-7M), and with the HCP1 with higher apparent affinity (KD= 10-8 M). The results indicate that the binding to citrullinated peptides is characterized by wide differences in affinity, with slower association and faster dissociation rate in the case of antibodies to viral citrullinated peptides, as compared to antibodies specific for the histone peptide. This biosensor analysis shows the high cross-reactivity of purified ACPA that bind other citrullinated peptides besides the one used for purification.