HUMAN ANTI-RO AUTOANTIBODIES BIND MULTIPLE CONFORMATIONAL EPITOPES OF60-KD RO AUTOANTIGEN

A gel filtration method was developed to estimate the number of confor mational epitopes on the 60-kD Ro antigen. Anti-Re Fab or Fab' was inc ubated with native Ro antigen at different ratios and the Stokes radiu s molecular weight of complexes was estimated by gel filtration. Bindi ng was saturated at 9 to 11 Fab molecules per bovine Ro molecule. Two additional Fab or Fab' were bound if human Ro was used as the antigen. Isolated Ro antigen/anti-Ro Fab complexes were evaluated for the rela tive proportion of antigen to antibody at saturation of antigen with a ntibody and thus stoichiometry was determined. This provided data supp orting there being between 7 and 11 binding sites, results similar to those with the gel filtration method. Experiments carried out with ant i-Re monoclonal antibodies showed one binding site per molecule of 60- kD Ro. Therefore, we have developed methods to count conformational ep itopes on autoantigens and have applied it to the Ro/anti-Ro system. T he data indicate that multiple conformational epitopes can be bound si multaneously by polyclonal anti-Re sera from patients with systemic lu pus erythematosus.