Transfection, overexpression and clinical application of human 60 kDa Ro/SSA autoantigens in HEp-2 cells.
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.
Lu
Liangjing
L
Shanghai Rheumatology Institute, and Department of Rheumatology, Renji Hospital of Shanghai Second Medical University, Shanghai 200001, China. luliangjing0@sina.com
Chen
Shunle
S
Gu
Yueying
Y
Shen
Nan
N
Bao
Chunde
C
Wang
Yuan
Y
Xue
Feng
F
Ye
Ping
P
Yu
Chongzhao
C
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
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