Enzyme-linked immunosorbent assay of anti-Ku antibodies using purified recombinant Ku antigens

Anti-Ku autoantibodies are frequently associated with scleroderma (PSS)-polymyositis (PM) overlap syndrome in Japanese patients. However, it has been also reported that anti-Ku antibodies are detected mostly in SLE patients in USA by enzyme-linked immunosorbent assay (ELISA) using a monoclonal anti-Ku antibody. To establish more sensitive detection system of anti-Ku antibodies, recombinant fusion proteins between Ku antigens and β-galactosidase expressed from isolated cDNA clones encoding the Ku proteins were purified and used them as antigens for ELISA. The 70kDa-Ku fusion protein (70kDa-Ku coupled with β-galactosidase: fp 70) and the 80kDa-Ku fusion protein (80kDa-Ku coupled with β-galactosidase: fp 80) were successfully purified through electro-elution from polyacrylamide gel slices without loss of immunoreactivity. New ELISA systems using these two fusion proteins were established and tested to detect the reactivity of sera from 126 patients with various systemic rheumatic diseases and normal healthy controls. Anti-Ku antibodies were detected in 77% and 46% of patients with overlap syndromes by fp 70-ELISA and fp 80-ELISA, respectively. Moreover, the sera from patients with PSS-PM overlap were revealed to contain the highest titers in both ELISA systems. Anti-Ku antibodies detected by both ELISAs were less frequent and with lower titers in patients with SLE, PSS, PM/DM and MCTD. ELISA using β-galactosidase as a control was weakly positive only in 5% of 126 patients and 20 normal healthy persons. These results indicate that ELISA using purified recombinant fusion proteins may be a good assay system for detecting anti-Ku autoantibodies.