Upon activation of the complement system by IgG immune aggregates several components become tightly bound to the aggregates. The covalent interaction of C3 with immune complexes is essential for the solubilization and inhibition of immune precipitation of the complexes. It has recently been reported that on erythrocytes that have a fixed complement, activated C3 can become involved in the formation of C3b-C3b covalent dimers, which acts as high-affinity binding sites for C5 (Kinoshita, T., Takata, Y., Kozono, H., Takeda, J., Hong, K. and Inoue, K., J. Immunol. 1988 141: 3895). To characterize the molecular composition of immune aggregates that have fixed complement by the alternative pathway, we have investigated whether such C3b-C3b dimers are formed in IgG immune complexes. For this purpose immune aggregates bearing covalently bound C3 were analyzed by two-dimensional gel electrophoresis and the resolved bands transferred to polyvinylidene difluoride membranes and sequenced. When immune aggregates were incubated with serum for 15 min at 37 degrees C, the major high-molecular mass bands detected by gel electrophoresis corresponded to heavy chain-C3 alpha 65 and C3 alpha 65-C3 alpha 43 (derived from iC3b-iC3b-IgG) covalent complexes. If K76COONa, an inhibitor of factor I, was added to the serum, before incubation with the immune complexes, then the major C3 alpha fragment detected on the complexes corresponded to the C3 alpha' chain (105 kDa) and not C3 alpha 65. Hence C3b-C3b covalent dimers are readily formed on the immune aggregates incubated with normal human serum, and are degraded to iC3b-iC3b by factor I. The second C3b molecule was shown to be bound to the C3 alpha 43 region (C-terminal portion of the C3 alpha' chain) of the first C3b molecule, which was itself covalently bound to the heavy chain of IgG. Covalent complexes of heavy chain-(C3 alpha 65)2 molecular composition were also detected, but their precise bonding pattern has not been established.

The measurement of anti-double-stranded DNA (anti-dsDNA) antibodies is a useful tool for the diagnosis and the follow-up of systemic lupus erythematosus (SLE). Anti-dsDNA antibodies are involved in the pathogenesis of lupus nephritis and they are, specially the high-avidity antibodies, the most specific antibodies associated with SLE nephritis and active SLE. The aim of the present study was to assess the clinical utility of an enzyme-linked immunosorbent assay (EUSA) that utilizes a circular double-stranded plasmid DNA as a nucleic acid source, adapted to an automated fluorescence immunoassay (EliA dsDNA, Pharmacia, Freiburg, Germany). Also, we compared this method with other immunoassays used in clinical laboratories. We have measured anti-dsDNA antibodies in the serum of 179 patients with a positive result for antinuclear antibodies (ANA). Seventy six sera were from SLE patients (14 men and 62 women), and the other 103 sera (from 20 men and 83 women) constituted the control group. This latter group includes nine Sjogren's syndrome patients, six patients with rheumatoid arthritis and 88 with various other diseases, including connective tissue diseases (n=34), hepatopathies (n= 17; 11 primary biliary cirrhosis and 6 autoimmune hepatitis), and 37 patients with nonautoimmune diseases (viral hepatitis, renal disease, diabetes, exanthema and hypertension). Methods used were "EliA dsDNA" (Pharmacia, Germany), "Varelisa dsDNA" (Pharmacia, Germany), Farr (Amersham, UK) and Chritidia luciliae immunofluorescence test (Vitro-Immun, Germany). We assessed sensitivity, specificity, positive predictive value and negative predictive value in the clinical study, and kappa index and scatter plots in the comparative study. The results show a low concordance between methods (kappa < 0.6). The evaluated EliA method shows a very good specificity for SLE (93.2%) and a good sensitivity for active SLE (70.8%).

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Ovalbumin-antiovalbumin IgG immune aggregates were incubated with normal human serum in the presence of iodo[1-14C]acetamide, in conditions in which only the alternative pathway of complement was activated. The [14C]C3b-IgG covalent complexes formed were digested with pepsin, and analysed by SDS/polyacrylamide-gel electrophoresis and fluorography. Covalent complexes of [14C]C3-Fd and [14C]C3-pFc' were visualized, demonstrating that, during complement activation by the alternative pathway, C3 is covalently incorporated into the C gamma 3 domain of IgG, as well as into the Fd region. The C gamma 2 domain becomes protected from pepsin action by the bound C3b. All the covalent linkages between C3 and the IgG were sensitive to hydroxylamine. When [14C]C3-pFc' covalent complexes were treated with 1 M-NH2OH and loaded onto a Bio-Gel P-4 column, a radioactive peak of 3 kDa was obtained. The material released from [14C]C3-pFc' and [14C]C3-F(ab')2 complexes after treatment with 1 M-NH2OH was mixed and analysed in the Bio-Gel P-4 column. A similar radioactive peak of 3 kDa was obtained. When this peak, either from [14C]C3-pFc' alone or from the mixture of [14C]C3-F(ab')2 and [14C]C3-pFc', was fractionated by h.p.l.c., virtually the same radioactive peptide profile was obtained, indicating that very similar C3 peptides remained covalently bound to both regions (Fab and C gamma 3) of the antibody molecule. It is suggested that C3 bound to the C gamma 3 domain of IgG may interfere with the Fc-Fc interactions of immune aggregates and thus may be involved in several biological properties displayed by these complement-activating aggregates.