Masataka Kuwana1, Kyoko Kimura, Yutaka Kawakami. 1. Institute for Advanced Medical Research, Keio University School of Medicine, 55 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. kuwanam@sc.itc.keio.ac.jp
Abstract
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%. CONCLUSION: 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.
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%. CONCLUSION: 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.