title: Detection of autoantibodies to SS-A/Ro by indirect immunofluorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp-2 cells.
abstract: The objective of the study was to determine the sensitivity and specificity of an indirect immunofluorescence (IIF) assay using transfected HEp-2 cells to detect anti-SS-A/Ro autoantibodies in human sera. Seventy-three sera having SS-A/Ro autoantibodies as determined by double immunodiffusion (ID) and immunoblotting (IB) were tested by IIF on a HEp-2 cell substrate that had been transfected with a full-length cDNA encoding a human 60 kD SS-A/Ro autoantigen. Controls included 30 normal human sera and 50 sera with a variety of other antinuclear antibodies. Prototype human and rabbit sera directed against the 60 kD SS-A/Ro antigen produced intense speckled nuclear and nucleolar staining of transfected cells. Sixty-nine of 73 (95%) SS-A/Ro positive sera also produced this characteristic staining pattern. The endpoint autoantibody titers on transfected cells was fivefold greater than on untransfected cells. The 30 normal human sera and the 50 sera with other antinuclear antibodies did not produce this characteristic staining. Six of 32 (19%) unselected sera that were sent for autoantibody testing had reactivity with transfectants by IIF. Four of the six sera were confirmed to have anti-SS-A/Ro antibodies by ID and 5/6 by IB. By contrast, only three of these sera were scored as having a staining pattern compatible with SS-A/Ro antibodies by IIF on standard HEp-2 substrates. We conclude that SS-A/Ro autoantibodies can be detected by an IIF assay using a HEp-2 cell substrate transfected with a SS-A/Ro cDNA. This new substrate detects SS-A/Ro antibodies that were not identified on standard HEp-2 substrates and by other immunoassays.
authors.affiliation: McCaig Center for Joint Injury and Arthritis Research, Faculty of Medicine, University of Calgary, Canada.
authors.forename: M J
authors.initials: MJ
authors.lastname: Fritzler
authors.forename: B J
authors.initials: BJ
authors.lastname: Miller
mesh_terms: D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology*; D018076:DNA, Complementary / Q000276:immunology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000276:immunology; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics / Q000276:immunology; D020733:RNA, Small Cytoplasmic; D015203:Reproducibility of Results; D012261:Ribonucleoproteins / Q000276:immunology*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity / Q000276:immunology; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000276:immunology; D000099207:SS-A Antigen
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D018076:DNA, Complementary; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human