Anti-Ro52 reactivity is an independent and additional serum marker in connective tissue disease

Abstract

Objective: To determine whether anti-Ro52 is an independent serum marker in connective tissue disease.

Methods: Over a two year period, 1727 consecutive antinuclear antibody (ANA) positive serum samples were analysed in parallel by double immunodiffusion with thymus/spleen nuclear extract and by line immunoassay with recombinant Ro52, recombinant La/SSB, and natural Ro60. Sera that were only reactive towards Ro52 were further analysed by a variety of additional anti-SSA/Ro detection methods and by specific anti-Ro52 and anti-Ro60 assays. Natural purified SSA/Ro was analysed by immunoblot and protein sequencing.

Results: Analysis of natural purified SSA/Ro (Immunovision, Springdale, AR) showed only Ro60 and no immunoreactive Ro52. Consequently, assays based on this substrate only identify sera with anti-Ro60 reactivity. Twenty serum samples showed anti-Ro52 without anti-Ro60 and anti-SSB/La on line immunoassay. By additional testing, 2/20 sera were found positive for anti-Ro60 reactivity. The remaining 18 sera were not identified by any of the classical anti-SSA/Ro assays and were considered to be reactive only with Ro52 and not with Ro60. This anti-Ro52 reactivity was confirmed by natural and recombinant Ro52 in 16/18 cases. 12/18 sera corresponded to connective tissue diseases.

Conclusion: Anti-Ro52 positive sera without any evidence of anti-Ro60 and anti-La/SSB reactivity can be considered as an independent group that is systematically missed by classical anti-SSA/Ro detection methods owing to a bias towards anti-Ro60 reactivity. The anti-Ro52 sera are precipitin negative, not retrieved by SSA/Ro enzyme linked immunosorbent assays (ELISAs) based on natural SSA/Ro, and show no specific ANA fluorescence staining pattern. These findings together with the clinical data indicate that anti-Ro52 should be considered as an additional and independent serum marker.

Figure 1

Analysis of natural purified SSA/Ro (Immunovision). No Ro52 was detected on immunoblot using different reagents. Reference serum with anti-Ro60 and high titre of anti-Ro52 antibodies (lane +). Control serum negative for anti-SSA (lane C). Blank control (lane B). Monoclonal anti-Ro60 antibody (lane mAb a-Ro60). Monoclonal anti-Ro52 antibody (lane mAb a-Ro52). Coomassie brilliant blue staining of natural purified SSA/Ro and protein sequencing of the 42 kDa band shows three peptides attributable to Ro60 (lane CBB). Reactivity of the monoclonal anti-Ro52 antibody in western immunoblot was confirmed (see fig 2, lane mAb a-Ro52).

Figure 2

Confirmation of anti-Ro52 reactivity by HeLa-S100 immunoblot. Four sera reactive with Ro52 on line immunoassay (Lane 10417, 8990, 10746, 14216) (top) without detectable anti-Ro60 and anti-La/SSB antibodies. Confirmation of this anti-Ro52 reactivity by HeLa-S100 immunoblot (bottom). Monoclonal anti-Ro60 antibody (lane mAb a-Ro60). Monoclonal anti-Ro52 antibody (lane mAb a-Ro52). Monoclonal anti-SSB antibody (lane mAb a-SSB).