DETECTION OF ANTI-DNA TOPOISOMERASE-I ANTIBODY BY AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY USING OVERLAPPING RECOMBINANT POLYPEPTIDES

Five recombinant fusion proteins with overlapping amino acid sequences encompassing the entire DNA topoisomerase I (topo I) molecule were ge nerated, purified, and used as antigens for an enzyme-linked immunosor bent assay (ELISA). IgG, IgA, IgM, and ''total'' (total of IgG, IgA, a nd IgM isotypes) anti-topo I antibodies were measured using a mixture of these five fusion proteins in 73 systemic sclerosis (SSc) sera posi tive for anti-topo I antibody by double immunodiffusion (DID) and 184 control sera negative for anti-topo I antibody by DID. Fragment-specif ic anti-topo I antibodies were also measured by ELISA using each topo I recombinant protein as antigen. IgG, IgA, IgM, and total anti-topo I antibodies were detected in 67 (92%), 56 (77%), 16 (22%), and 70 (96% ) of 73 SSe sera positive for anti-topo I antibody by DID, respectivel y. The specificity of the total anti-topo I ELISA was 99% when compare d with DID. The total anti-topo I ELISA levels were strongly correlate d with DID titers (r = 0.907, P < 0.0001). Three sera which recognized a conformational epitope on native topo I or had predominantly IgM an ti-topo I antibody showed a false-negative result with the total anti- topo I ELISA. Three SSe sera negative for anti-topo I antibody by DID were positive by the total anti-topo P ELISA, and two were confirmed t o recognize the N-terminus of topo I. IgG and IgA antibody levels to t he N-terminal and central portion of topo I were correlated with each other, but those to the C-terminus were not. These findings indicate t hat the ELISA using recombinant fusion proteins is a highly sensitive and specific alternative to conventional DID for the detection of anti -topo I antibody. (C) 1995 Academic Press, Inc.