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Abstract
Background Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and mediate pathogenesis via the formation of immune complexes. While the avidity of these antibodies is high, it depends on monogamous bivalency, a mode of antibody binding in which both IgG combining sites interact with an extended piece of DNA. In the current study, we investigated this interaction further by assessing the activity of Fab and F(ab’)2 preparations of IgG from plasmas of SLE patients.
Materials and methods Using purified IgG, Fab fragments were generated by papain digestion while F(ab’)2 fragments were prepared with pepsin. The binding to native calf thymus (CT) DNA was assessed by ELISA using an anti-human IgG (Fab specific) peroxidase reagent. In these experiments, the concentrations of IgG and fragments were determined on the basis of an equivalent number of binding sites. Control antigens were tetanus and an EBV antigen preparation. IgG and fragments from normal human subjects were used as controls for binding to foreign antigens.
Results For each of the SLE IgG preparations studied, Fab and F(ab’)2 fragments failed to bind significantly to DNA in the ELISA (Figure 1). In contrast, the Fab and F(ab’)2 fragments were active against the tetanus and EBV antigens. The binding of the fragments from SLE patients to the foreign antigens was similar to that of normal human subjects.
Conclusions These results define a new pattern of anti-DNA binding. Since a Fab fragment can bind monovalently, a lack of activity is expected. The failure of F(ab’)2 fragments to bind is unexpected, pointing to a critical role for the Fc portion of the IgG molecule in stabilising antibody interaction. The Fc portion can contribute to anti-DNA by inducing a conformational change in the binding sites; contacting DNA; or forming Fc:Fc interactions to increase valency. This binding pattern can be called Fc-dependent monogamous bivalency. The findings suggest that agents that affect the Fc portion may be useful to therapeutically inhibit anti-DNA interactions.
The binding of intact IgG and F(ab')2 fragments to DNA was determined by ELISA using calf thymus DNA as antigen.