Blotting patterns of IgG anti-(U1)RNP antibodies in mixed connective tissue disease

Abstract

Serum reactivities towards individual U1 snRNP proteins were determined by immunoblotting in 32 patients with mixed connective tissue disease (MCTD). Time persistence of immunoblot profiles and clinical significance of anti-(U1)RNP antibody specificities were also investigated. IgG anti-(U1)RNP antibodies were found in the sera of 29 out of 32 patients (90.6%): 21 (65.6%) reacted with the 70-kD protein, 25 (78.1%) with A, 23 (71.9%) with C and 20 (62.5%) with B/B' proteins. None were reactive with the Sm-D peptide. Seventy kilodalton antibody specificity was strongly associated with a higher antinuclear antibody titre (> 160) and slightly associated with disease activity; anti-B/B' specificity was associated with lymphadenopathy. Anti-A, -C and -B/B' antibodies were negatively associated with systemic lupus erythematosus (SLE) skin rashes. Two types of anti-(U1)RNP blotting patterns were selected: "full spectrum" (53.1% of cases) and a "partially/no reactive" one (46.9%). Such patterns were unchanged over time in 14 out of 16 cases prospectively examined (87.5%), while the pattern shifted from "full spectrum" to "partially/no reactive" in 2 cases (12.5%): in 1 after a prolonged clinical remission (> or = 4 years) and in the other following immunosuppressive therapy. The anti-(U1)RNP antibody immunoblot profile in MCTD patients consisted of various reactivities and remained unchanged over time in most cases. Antibody reactivity against the 70-kD protein represented the major U1 snRNP specificity. The various anti-(U1)RNP specific reactivities demonstrated poor clinical significance within MCTD. Thus, MCTD seems to be characterized by a longstanding serological heterogeneity whose reactivities do not apparently correspond to distinct features within the broad clinical spectrum of MCTD.

The appearance of U1 RNP antibody specificities in sequential autoimmune human antisera follows a characteristic order that implicates the U1-70 kd and B?/B proteins as predominant U1 RNP immunogens

“…In contrast to prior studies that have examined the longitudinal evolution of RNP immunity in human series (17–21), the cohort in our study was not preselected to already have RNP immunity. Thus, we could observe the order in which initial seroconversion to anti‐RNP antibodies developed.…”

Anti-ribosomal P protein antibodies detected by immunoblotting in patients with connective tissue diseases: their specificity for SLE and association with IgG anticardiolipin antibodies

“…Anti-small nuclear ribonucleoproteins (RNPs) and other anti-extractable nuclear antigen antibodies were detected by counterimmunoelectrophoresis28 and confirmed by immunoblotting on nuclear and cytoplasmic Raji cells extracts, resolved on 12.5% and 15% SDS-PAGE respectively, by the methods mentioned above 2227 Antinuclear antibodies, anticentromere and anti-double stranded (ds) DNA antibodies were detected by indirect immunofluorescence on Hep-2 cells and Crithidia luciliae , respectively.…”

“…IgG antibodies to ribosomal P proteins and to other cytoplasmic antigens were detected by western blotting as previously described 22. Briefly, nuclear and cytoplasmic extracts were prepared from Raji cells cultured in RPMI 1640 containing 10% heat inactivated fetal calf serum (Gibco UK, Ltd), penicillin, and streptomycin, according to McHugh et al 23.…”

High serum levels of antibodies against the recombinant 70 kDa ribonucleoprotein are useful for diagnosing mixed connective tissue disease

“…Although commercially available ELISA tests are able to measure Sm and U1‐RNP with reasonable accuracy 20,21 they are not well suited for differentiating MCTD and SLE as these ELISA tests usually contain mixtures of the U 1 ‐snRNP and of the Sm‐proteins as antigenic substrates. A cross‐reactive epitope motif is found both on the A and C proteins in U 1 ‐snRNP and on the SmBB′ protein, resulting in false‐positive detection of anti‐ U 1 ‐snRNP antibodies in up to 40% of SLE sera and false‐positive detection of anti‐Sm antibodies in MCTD sera 7,10 …”

“…Although anti‐Sm antibodies can react with at least nine different proteins (B, B′, N, D1, D2, D3, E, F and G) that are shared by all members of the U‐snRNP family, the autoimmune reaction in SLE is usually directed against the BB′ and D proteins 7–9 . However, the presence of anti‐Sm antibodies does not distinguish SLE from MCTD, as positive reactions to Sm antigens have also been described in MCTD sera 7,10 due to a cross‐reactivity between the BB′ subset of Sm antigen and the U 1 ‐snRNP molecule.…”