title: Detection of anti-DNA topoisomerase I antibody by an enzyme-linked immunosorbent assay using overlapping recombinant polypeptides.
abstract: Five recombinant fusion proteins with overlapping amino acid sequences encompassing the entire DNA topoisomerase I (topo I) molecule were generated, purified, and used as antigens for an enzyme-linked immunosorbent assay (ELISA). IgG, IgA, IgM, and "total" (total of IgG, IgA, and IgM isotypes) anti-topo I antibodies were measured using a mixture of these five fusion proteins in 73 systemic sclerosis (SSc) sera positive for anti-topo I antibody by double immunodiffusion (DID) and 184 control sera negative for anti-topo I antibody by DID. Fragment-specific 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 SSc sera positive for anti-topo I antibody by DID, respectively. The specificity of the total anti-topo I ELISA was 99% when compared with DID. The total anti-topo I ELISA levels were strongly correlated with DID titers (r = 0.907, P < 0.0001). Three sera which recognized a conformational epitope on native topo I or had predominantly IgM anti-topo I antibody showed a false-negative result with the total anti-topo I ELISA. Three SSc sera negative for anti-topo I antibody by DID were positive by the total anti-topo I ELISA, and two were confirmed to recognize the N-terminus of topo I. IgG and IgA antibody levels to the N-terminal and central portion of topo I were correlated with each other, but those to the C-terminus were not. These findings indicate that the ELISA using recombinant fusion proteins is a highly sensitive and specific alternative to conventional DID for the detection of anti-topo I antibody.
authors.affiliation: Department of Medicine, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
authors.forename: M
authors.initials: M
authors.lastname: Kuwana
authors.forename: T A
authors.initials: TA
authors.lastname: Medsger
authors.forename: T M
authors.initials: TM
authors.lastname: Wright
mesh_terms: D000328:Adult; D000368:Aged; D000974:Antibodies, Antinuclear / Q000145:classification / Q000276:immunology*; D004264:DNA Topoisomerases, Type I / Q000276:immunology* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006367:HeLa Cells; D006801:Humans; D005779:Immunodiffusion; D007070:Immunoglobulin A / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis; D007132:Immunoglobulin Isotypes / Q000097:blood; D007075:Immunoglobulin M / Q000032:analysis; D008297:Male; D008875:Middle Aged; D010455:Peptides; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
chemical_list: D000974:Antibodies, Antinuclear; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004264:DNA Topoisomerases, Type I