Identification of an immunodominant epitope on RNA polymerase III recognized by systemic sclerosis sera: Application to enzyme‐linked immunosorbent assay

Publisher Website
Google Scholar
Abstract
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. 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. 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%. 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.