Human anti-Ro autoantibodies bind multiple conformational epitopes of 60-kD Ro autoantigen.
A gel filtration method was developed to estimate the number of conformational epitopes on the 60-kD Ro antigen. Anti-Ro Fab or Fab' was incubated with native Ro antigen at different ratios and the Stokes radius molecular weight of complexes was estimated by gel filtration. Binding 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 relative proportion of antigen to antibody at saturation of antigen with antibody and thus stoichiometry was determined. This provided data supporting there being between 7 and 11 binding sites, results similar to those with the gel filtration method. Experiments carried out with anti-Ro monoclonal antibodies showed one binding site per molecule of 60-kD Ro. Therefore, we have developed methods to count conformational epitopes on autoantigens and have applied it to the Ro/anti-Ro system. The data indicate that multiple conformational epitopes can be bound simultaneously by polyclonal anti-Ro sera from patients with systemic lupus erythematosus.
Huang
S C
SC
Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Scofield
R H
RH
Kurien
B T
BT
Harley
J B
JB
D000818:Animals; D000974:Antibodies, Antinuclear / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000378:metabolism; D001324:Autoantigens / Q000276:immunology*; D001666:Binding Sites, Antibody; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D008970:Molecular Weight; D011487:Protein Conformation; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins / Q000276:immunology*; D000099207:SS-A Antigen
D000974:Antibodies, Antinuclear; D000936:Antigen-Antibody Complex; D001324:Autoantigens; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human; C035356:SS-A antibodies