Transfection of mammalian cells with nuclear autoantigens: The new approach to diagnosing ANA

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Abstract

The IF-ANA is a valuable screening test for the presence of an autoimmune rheumatic disease but has lower diagnostic specificity than the ENA. Furthermore, the ANA may be negative or only weakly positive with low abundance autoantigens such as Ro and Jo-1. Detection of anti-Ro poses special problems: CIE (the gold standard for anti-Ro) can be technically difficult; Western blotting has poor sensitivity; and ELISAs can give false positives and negatives. Ro60, Ro52 and La are the most common ENA specificities encountered in the diagnostic laboratory and of considerable clinical importance. Full-length cDNAs encoding these three genetically distinct proteins have been transfected into and over-expressed in the human cell line HEp-2, and their utility as ANA substrates examined. In transfected murine cell lines, human La was expressed as a predominantly nuclear antigen with a typical pattern of nuclear speckling. The human molecule was functionally conserved across species, associating with murine Ro60 and binding to RNAs. The sensitivity of anti-La detection was not increased in the La HEp-2 transfectants. This was not unexpected since La is an abundant nuclear antigen. However, Ro60 HEp-2 transfectants showed remarkably increased sensitivity for anti-Ro without loss of specificity. Characteristic nucleoli and nuclear staining of these transfectants allowed a rapid diagnosis of anti-Ro which correlated strongly with CIE. Rp52 is a recently characterised Ro antigen which is structurally linked to Ro60 and La. High titre anti-Ro52 antibodies are associated with neonatal lupus erythematosus and congenital heart block. The Ro52 HEp-2 transfectants showed diffuse staining of the cytoplasm indicating a requirement for nuclear localisation signals. These cells were of less diagnostic utility compared with the Ro60 transfectants; recombinant Ro52 ELISA was found to be the most sensitive and simple method for detection of anti-Ro52 autoantibodies in the diagnostic laboratory. Transfection techniques are likely to be developed for a number of autoantibody systems and will increase the turnaround time of ANA/ENA testing. The mixture of transfected cells and Ro60 transfectants is a particularly useful IF-ANA screening substrate, since anti-Ro can be readily detected without loss of other ANA specificities.

Original languageEnglish
Pages (from-to)156-157
Number of pages2
JournalAustralian Journal of Medical Science
Volume17
Issue number4
Publication statusPublished - 1 Dec 1996
Externally publishedYes

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