Detection of anti-B/B' UsnRNP antibodies in connective tissue disease sera by Western immunoblot

Introduction

The fine antibody specificity towards protein components of uridine-enriched small nuclear ribonucleoproteins (UsnRNP) may be investigated by several methods including the Western immunoblot. Crucial in Western blot techniques' reliability is the origin and nature of the antigenic source.

Aim

To assess the significance of antibodies to B/B' proteins detected by Western immunoblot in connective tissue disease (CTD) patients.

Methods

Three hundred and forty-eight patients with well diagnosed CTD (101 SLE, 51 systemic sclerosis, 53 primary Sjogren's syndrome, 27 poly/dermatomyositis, 15 rheumatoid arthritis and 101 overlap CTD) and 31 matched healthy subjects were studied. In addition, sera from 13 patients with primary Epstein-Barr virus (EBV) infection (10 in acute primary infection and 3 with anamnestic past infection) and high titer IgG anti-EBV antibodies were tested. IgG anti-UsnRNP as well as anti-ribosomal P protein antibodies were determined by Western blotting on total Raji cell extract (a cell line transformed by EBV). Antinuclear and anti-dsDNA antibodies were detected by indirect immunofluorescence on HEp-2 cells and Crithidia luciliae respectively, anti-ENA by counterimmunoelectrophoresis. Statistical analysis was performed by chi-square test.

Results

An unespectedly high frequency of anti-B/B' antibodies was found, confined to SLE (54.4%) and overlap CTD with SLE features (55.2%). Anti-B/B' antibodies were closely associated with other anti-UsnRNP antibodies (P < 0.0001), gel precipitating anti-nRNP antibodies (P < 0.0001) and anti-ribosomal P antibodies (P = 0.0013). Band patterns unequivocally different from those obtained with autoimmune sera, were provided by anti-EBV positive sera. Noteworthy, a peptide with an apparent MW corresponding to that of B peptide (28 kDa) was clearly recognized by 9/10 sera from active EBV infection but not by anamnestic EBV infection sera.

Conclusions

The Sm spliceosomal complex is one of the most important targeted autoantigens in SLE. Western immunoblot on Raji cells provides a reliably sensitive and specific antigenic source for anti-Sm B/B' antibodies. Such high immunoreactivity could be explained by the strong cross-reactive potential of B/B' proteins and not by EBV cell transformation. Further studies are in progress to comparatively evaluate the suitability of other cell lines as an antigenic source.