Kinetics of inter-heavy chain disulfide bond formation of liganded and unliganded human immunoglobulin G by radioimmunoassay.

Monoclonal antibodies (mAbs) to the hinge region of human IgG1 immunoglobulins were prepared by immunization with a proteolytic fragment of hinge segment coupled to keyhole limpet hemocyanin. A mAb, 4G3, was obtained capable of binding to intact IgG but not to partially reduced IgG. Using this mAb, inter-heavy (H) chain disulfide bond formation from partially reduced anti-tetanus antibodies (Abs) in the presence or absence of antigens (Ags) was studied by solid phase radioimmunoassay. When the Abs were partially reduced in solution and then coated to plastic plates, only 10% regeneration of inter-H chain disulfide bonds occurred after reoxidation, although 100% formation occurred in solution [Kishida et al., J. Biochem. (Tokyo) 79, 91-105 (1976)]. This difference in the extent of disulfide bond formation can be explained by the fact that there are two convertible isomers in solution, Conformer I and II, one of which (Conformer I) can form disulfide bonds but is present as a minor component. Since the motion of IgG molecules on plates is restricted by hydrophobic interactions, the two conformers are not convertible as in solution. Therefore only Conformer I which existed before coating formed inter-H chain disulfide bonds. Similar kinetic measurements were performed using plates coated with tetanus toxoid. Abs, partially reduced in solution and then allowed to react with Ags on the plate were able to completely regenerate their inter-H chain disulfide bonds, although the rate of reaction was slow. These results can also be explained by the fact that the two isomers are convertible since Ag-Ab complexes are dissociable. Ag-binding therefore did not significantly perturb conformation of the hinge segments. In addition, no difference between liganded and unliganded Abs was observed in the binding of anti-hinge mAbs. These results imply little or no contribution of the hinge region to transmission of the signal produced by Ag-binding to Fc.