Recombinant ribosomal P2 protein can unmask anti-ribosomal P autoantibodies from healthy adults.
Autoantibodies to ribosomal P proteins (anti-P) are detected almost exclusively in the serum samples from patients with systemic lupus erythematosus when conventional enzyme-linked immunosorbent assay and immunoblotting techniques are used. Anti-P are not detected in serum samples from healthy adults by these techniques. By treating serum from healthy adults with ribosome-coated beads, we unexpectedly unmasked anti-P in virtually all individuals. This unmasking of anti-P occurs by the displacement of an antibody inhibitor from anti-P. We wanted to determine whether anti-P from healthy adults could also be unmasked by treatment of their serum or plasma with isolated ribosomal P proteins. Recombinant human ribosomal P2 protein was produced in bacteria as a TrpE fusion protein, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and isolated as strips of membranes corresponding to the size of the P2 fusion protein. Serum or plasma from six healthy adults and three patients with systemic lupus erythematosus were incubated with these strips overnight rather than for 2 hours, as is done in conventional immunoblots. Acid eluates were obtained from the strips and analyzed for antibody activity by immunoblot. Eluates from healthy adults and patients contained antibodies reactive with recombinant ribosomal P2 protein. They also reacted with the three ribosomal P proteins in purified rabbit ribosomes. Their anti-P activity was completely inhibited by a peptide corresponding to the immunodominant linear epitope of the ribosomal P proteins. The antibodies in the eluate were immunoglobulin G. We conclude that anti-P autoantibodies from healthy adults can be unmasked by affinity purification on denatured, recombinant ribosomal P proteins and that antigen excess is sufficient for inhibitor displacement.
Pan
Z J
ZJ
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Stafford
H A
HA
D000328:Adult; D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D016056:Immunodominant Epitopes; D007074:Immunoglobulin G / Q000097:blood; D007132:Immunoglobulin Isotypes / Q000097:blood; D008297:Male; D010750:Phosphoproteins / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012269:Ribosomal Proteins
D001323:Autoantibodies; D016056:Immunodominant Epitopes; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C031937:phosphoprotein P2, ribosomal