Transfection, overexpression and clinical application of human 60 kDa Ro/SSA autoantigens in HEp-2 cells / 中华医学杂志(英文版)
Chinese Medical Journal
;
(24): 350-353, 2003.
Article
in English
| WPRIM
| ID: wpr-356799
ABSTRACT
<p><b>OBJECTIVE</b>To develop an improved substrate for indirect immunofluorescence test (IIF) for detecting anti-Ro60/Sjogren's syndrome A (Ro/SSA) autoantibodies.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Ribonucleoproteins
/
Autoantigens
/
Blood
/
Recombinant Fusion Proteins
/
Transfection
/
Cell Line
/
Antibodies, Antinuclear
/
Fluorescent Antibody Technique, Indirect
/
RNA, Small Cytoplasmic
/
Allergy and Immunology
Limits:
Humans
Language:
English
Journal:
Chinese Medical Journal
Year:
2003
Type:
Article
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