title: Transfection and overexpression of the human 60-kDa Ro/SS-A autoantigen in HEp-2 cells.
abstract: A full-length cDNA encoding human 60-kDa Ro/SS-A protein was transfected into and overexpressed in the human cell line HEp-2, with the aim of developing an improved reagent for indirect immunofluorescence (IF) detection of anti-Ro autoantibodies. Stable transfectants were analyzed by IF using a panel of 20 precipitin-positive anti-Ro human sera. Transfectants showed bright finely speckled nuclear and nucleolar staining. No surface membrane expression was detected despite marked overexpression of 60-kDa Ro. All human anti-Ro sera reacted with the transfectants with titers ranging from 1:320 to 1:40,960. The same sera tested on untransfected cells showed titers from negative (3 sera) to 1:160. These transfectants dramatically increase the sensitivity of IF testing (mean increase in titer of 41-fold) and allow detection of specific anti-Ro antibodies in samples negative or equivocal on untransfected cells. The staining patterns of antisera of other important specificities remained unaltered. In a study of sera from 22 patients with systemic lupus erythematosus, anti-60-kDa Ro autoantibodies were detected in all sera when tested on 60-kDa Ro-transfected HEp-2 cells; however, in 12 of 22 sera autoantibodies were undetectable by recombinant 60-kDa Ro ELISA. The 60-kDa Ro transfectants are a simple and sensitive method for detection of anti-Ro antibodies in patients with systemic rheumatic diseases.
authors.affiliation: Department of Clinical Immunology, Flinders Medical Centre, Bedford Park, Australia.
authors.forename: C L
authors.initials: CL
authors.lastname: Keech
authors.forename: J
authors.initials: J
authors.lastname: McCluskey
authors.forename: T P
authors.initials: TP
authors.lastname: Gordon
mesh_terms: D001323:Autoantibodies / Q000032:analysis; D001324:Autoantigens / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007822:Laryngeal Neoplasms; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000276:immunology; D000099207:SS-A Antigen
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human