Clinical relevance and HLA association of autoantibodies against the nucleolus organizer region (NOR-90).

OBJECTIVE: NOR-90 autoantibodies directed against the nucleolus organizer region (NOR) have been described as rare scleroderma associated antibodies. We studied the clinical features of patients with NOR-90 antibodies as well as their HLA phenotype. METHODS: NOR-90 antibodies were detected by indirect immunofluorescence assay using HEp-2 cells, by chromosome spreads as a substrate and in addition by Western blot analysis with HeLa-S3 nucleolar extract. HLA antigens of the NOR-90 antibody positive patients were typed with the standard NIH complement dependent microcytotoxicity test. RESULTS: Nine sera selected by means of the indirect immunofluorescence revealed a typical double band pattern of about 90 kDa identical with the pattern of 2 NOR-90 reference sera by Western blot analysis. Only one patient positive for NOR-90 antibodies suffered from systemic sclerosis (limited cutaneous scleroderma). The other patients with NOR-90 antibodies showed no signs of systemic sclerosis. All patients with NOR-90 antibodies were women and 8 of 9 patients (89 versus 13% of healthy controls, Pcorr < 0.001) were positive for the HLA-DR1 allele. CONCLUSION: In contrast to the first report on NOR-90 antibodies we demonstrated no association of these antibodies with systemic sclerosis; however, we found strong evidence for an immunogenetic background of NOR-90 antibody formation.

On the interaction of the first complement component C1 and its subunit C1q with solid-phase IgM immune complexes.

The interaction of C1 and C1q with solid-phase anti-dextran MOPC-104E IgM was studied. An enzyme-linked immunosorbent assay (ELISA) was used to detect bound C1q. The results revealed that immobilized IgM is converted to the functionally active 'staple' conformation by the specific polyvalent ligand dextran (B 1355/S). C1q is fixed to IgM dependent on the antigen concentration, and its binding might be explained by assuming a functional binding constant (K) of approximately 10(9) M-1. Molecules bound with a K in the range of 10(7) M-1 cannot be detected by this ELISA procedure. The fixation of C1q saturated with an excess of the C1r2S2-tetramer differs from that of free C1q. C1q incorporated in the C1 complex rapidly dissociates independently of the antigen concentration. Since the complement binding sites are located at definite positions on the IgM molecule because of its pentameric structure, it is suggested that the distinguishable association properties of C1 and C1q are brought about from the altered flexibility of the C1q molecule complexed with C1r2S2.

Failure of oligosaccharide MOPC-104E IgM complexes to bind C1q and to activate C1.

The capacity of anti-dextran MOPC-104E IgM to bind and activate the first complement component (C1) in the presence of various specific monovalent oligosaccharides was investigated. Enzyme-linked immunosorbent assay revealed that IgM-oligosaccharide complexes saturated up to 97% with ligands were not capable of binding C1q under physiological conditions. Nor was any activation of reconstituted C1 observed. These results indicate that occupation of the single IgM binding sites by a monovalent ligand is not sufficient to induce a signal for complement activation.

Human C1q: rapid isolation and quantitative determination by immunodiffusion.

A simple and rapid 2-step procedure for isolating C1q from human plasma at high yields (about 50%) is described. The purification involves diaminopropane precipitation followed by chromatography on IgG-Sepharose. The final product (obtained at a concentration of about 1.5 mg/ml) was electrophoretically and immunochemically pure and stable at -70 degrees C for long periods. The Mancini technique for the quantitative determination of C1q was reinvestigated and the use of gels containing high salt concentrations (1.0 M NaCl) was found to be absolutely necessary. A value of 0.076 mg/ml C1q in pooled human plasma was obtained.