Anti-citrullinated protein/peptide antibodies (ACPAs) 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-viral citrullinated peptide 1 (VCP1) and VCP2 
derived from Epstein-Barr virus (EBV)-encoded proteins and histone citrullinated 
peptide 1 (HCP1) derived from histone H4-were synthesized as tetrameric multiple 
antigen peptides and immobilized on sensor chips CM5 type in a Biacore T100 
instrument. Specific binding of purified antibodies from RA patients to the 
three peptides was analyzed by surface plasmon resonance using two 
arginine-containing sequences as controls. Employing a 1:1 binding model for 
affinity constant calculation, ACPAs interacted with VCP1 and VCP2 with lower 
apparent affinity (10(-6) M>KD>10(-7) M) and interacted with 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 rates in the case of antibodies to 
viral citrullinated peptides as compared with antibodies specific for the 
histone peptide. This biosensor analysis shows the high cross-reactivity of 
purified ACPAs that bind other citrullinated peptides besides the one used for 
purification.