title: Transfection, overexpression and clinical application of human 60 kDa Ro/SSA autoantigens in HEp-2 cells.
abstract: OBJECTIVE
  To develop an improved substrate for indirect immunofluorescence test (IIF) for detecting anti-Ro60/Sjogren's syndrome A (Ro/SSA) autoantibodies.
  
  
  METHODS
  60-kDa Ro/SSA autoantigens (Ro60) cDNAs were obtained from human placental cDNA library using polymerase chain reaction (PCR) and were cloned into the mammalian expression vector-pEGFP-C1. Then, the recombinant plasmids were transfected into HEp-2 cells. We confirmed the overexpression, localization and antigenicity of fusion proteins in transfected cells by means of immunoblotting, confocal fluorescence microscopy and IIF. HEp-2 and HEp-Ro60 were analyzed by IIF using a panel of 10 precipitin-positive anti-Ro human sera simultaneously.
  
  
  RESULTS
  Stable expression of Ro60-green fluorescent protein (Ro60-GFP) fusion proteins were maintained ten more generations. Ro60-GFP kept the antigenicity of Ro while demonstrating its own characteristic immunofluorescent pattern in HEp-Ro60 cells. The transfectants dramatically increased the sensitivity of IIF testing (a mean increase of 6.7-fold in endpoint titer). Eight over ten (8/10) positive anti-Ro sera showed characteristic immunofluorescent patterns for HEp-Ro60, including two sera that were anti-nuclear antibodies (ANA) negative for untransfected HEp-2. IIF-ANA in all healthy sera was negative for HEp-Ro60.
  
  
  CONCLUSIONS
  As a new substrate for IIF, the Ro60 transfectants can be used to detect anti-Ro antibodies. In addition, transfected HEp-2 cells keep the immunofluorescent properties of HEp-2 cells in IIF-ANA tests and can be employed as a substrate for routine IIF-ANA detection.
authors.affiliation: Shanghai Rheumatology Institute, and Department of Rheumatology, Renji Hospital of Shanghai Second Medical University, Shanghai 200001, China. luliangjing0@sina.com
authors.forename: Liangjing
authors.initials: L
authors.lastname: Lu
authors.forename: Shunle
authors.initials: S
authors.lastname: Chen
authors.forename: Yueying
authors.initials: Y
authors.lastname: Gu
authors.forename: Nan
authors.initials: N
authors.lastname: Shen
authors.forename: Chunde
authors.initials: C
authors.lastname: Bao
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Xue
authors.forename: Ping
authors.initials: P
authors.lastname: Ye
authors.forename: Chongzhao
authors.initials: C
authors.lastname: Yu
mesh_terms: D000974:Antibodies, Antinuclear / Q000097:blood*; D001324:Autoantigens; D002460:Cell Line; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008970:Molecular Weight; D020733:RNA, Small Cytoplasmic; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D012261:Ribonucleoproteins / Q000276:immunology*; D014162:Transfection; D000099207:SS-A Antigen
chemical_list: D000974:Antibodies, Antinuclear; D001324:Autoantigens; D020733:RNA, Small Cytoplasmic; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human; C035356:SS-A antibodies