Identification of an immunodominant epitope on RNA polymerase III recognized by systemic sclerosis sera: application to enzyme-linked immunosorbent assay.
OBJECTIVE
To characterize an immunodominant epitope on RNA polymerase III (RNAP III) recognized by systemic sclerosis (SSc) sera and to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of serum anti-RNAP I/III antibodies.


METHODS
RNAP III-specific subunits RPC62 and RPC155 were generated in a bacterial expression system as a series of recombinant fragments. Reactivities to these recombinant fragments were examined by immunoblots and/or ELISA in 16 SSc sera containing anti-RNAP I/III antibodies, 89 SSc sera lacking anti-RNAP I/III antibodies, 61 systemic lupus erythematosus (SLE) sera, and 61 healthy control sera.


RESULTS
Anti-RNAP I/III-positive SSc sera recognized several distinct epitopes on RPC62 and RPC155 in various combinations, but the fragment encoding amino acids at positions 732-1166 of RPC155 was recognized by all 11 anti-RNAP I/III-positive SSc sera tested. Carboxyl- and amino-terminal deletion studies showed that at least 130 amino acids at positions 891-1020 of RPC155 were necessary for the antibody binding, but strong reactivity required an additional amino-terminal extension. When a purified recombinant fragment containing the immunodominant epitope was used as the antigen source in an ELISA, elevated antibody reactivity was detected in all 16 anti-RNAP I/III-positive SSc sera, but in no anti-RNAP I/III-negative SSc, SLE, or healthy control sera, representing a sensitivity of 100% and a specificity of 100%.


CONCLUSIONS
A major epitope commonly recognized by SSc sera containing anti-RNAP I/III antibodies was identified on RPC155. The ELISA using a recombinant fragment expressing the immunodominant epitope should be a valuable tool for routine screening for anti-RNAP I/III antibodies in clinical diagnostic laboratories.
Kuwana
Masataka
M
Institute for Advanced Medical Research, Keio University School of Medicine, 55 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. kuwanam@sc.itc.keio.ac.jp
Kimura
Kyoko
K
Kawakami
Yutaka
Y
D001323:Autoantibodies / Q000276:immunology; D018076:DNA, Complementary / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D016056:Immunodominant Epitopes / Q000032:analysis* / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D012320:RNA Polymerase III / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D012595:Scleroderma, Systemic / Q000276:immunology*
D001323:Autoantibodies; D018076:DNA, Complementary; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C000610570:POLR3C protein, human; D012320:RNA Polymerase III