Characterization of continuous monoclonal antibody epitopes in the N-terminus of Ro60.

ABSTRACT

One of the major targets of the autoimmune response in the rheumatic autoimmune diseases, Systemic Lupus Erythematosus and Sjögrens Syndrome, is the protein Ro60. Ro60 is known to associate with small misfolded RNAs, and is involved in RNA quality control and in enhancing cell survival during cellular stress, e.g. after ultaviolet irradiation. In this study, six monoclonal antibodies to Ro60 were analyzed in order to identify antigenic regions and the nature of these. Preliminary analyses revealed that two of the antibodies recognized continuous epitopes, while the remaining antibodies most likely recognized conformational epitopes. The continuous epitopes of Ro60 were characterised by modified immunoassays employing resin-bound peptides and free peptides. Peptide screenings located the epitopes to the N-terminus of Ro60, and further analyses indicated that the epitopes of the monoclonal antibodies TROVE2 and SSI-HYB 358-02 were located to amino acids 8-17 and 34-49, respectively. Moreover, charged amino acids were found to be especially important for antibody reactivity, although antibody reactivity of the monoclonal antibody TROVE2 primarily was found to be epitope backbone-dependent.