title: Biosensor analysis of anti-citrullinated protein/peptide antibody affinity.
abstract: 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.
authors.affiliation: Laboratory of Peptide and Protein Chemistry and Biology, Department of NeuroFarBa, Division of Pharmaceutical Sciences and Nutraceutic, University of Florence, 50019 Florence, Italy.
authors.forename: Giada
authors.initials: G
authors.lastname: Rossi
authors.affiliation: Toscana Biomarkers, 53100 Siena, Italy.
authors.forename: Feliciana
authors.initials: F
authors.lastname: Real-Fernández
authors.affiliation: Clinical Immunology and Allergy Unit, Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy.
authors.forename: Filomena
authors.initials: F
authors.lastname: Panza
authors.affiliation: Toscana Biomarkers, 53100 Siena, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Barbetti
authors.affiliation: Clinical Immunology and Allergy Unit, Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Pratesi
authors.affiliation: Laboratory of Peptide and Protein Chemistry and Biology, Department of NeuroFarBa, Division of Pharmaceutical Sciences and Nutraceutic, University of Florence, 50019 Florence, Italy; Toscana Biomarkers, 53100 Siena, Italy. Electronic address: paolo.rovero@unifi.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Rovero
authors.affiliation: Toscana Biomarkers, 53100 Siena, Italy; Clinical Immunology and Allergy Unit, Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy. Electronic address: paola.migliorini@med.unipi.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Migliorini
mesh_terms: D000914:Antibodies, Viral / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity; D001681:Biological Assay / Q000379:methods*; D003429:Cross Reactions; D004854:Herpesvirus 4, Human / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008956:Models, Chemical; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D014764:Viral Proteins / Q000032:analysis* / Q000737:chemistry
chemical_list: D000914:Antibodies, Viral; C576376:EBNA 1 protein (35-58), citrullinated-; C569106:EBNA-2 protein (338-358), citrullinated-; D010446:Peptide Fragments; D014764:Viral Proteins
keywords: Affinity; Anti-citrullinated protein/peptide antibodies; Autoantibodies; Rheumatoid arthritis; Surface plasmon resonance