Appearance of Neoantigen in Mouse IgG1 Upon Reduction of Interchain Disulfide Bridges: Assessment of Local and General Conformational Rearrangements Using Monoclonal Antibody and Small-angle X-ray Scattering

Abstract

Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)2 fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between CH and CL domains. The second produced monAb F6C2 reacts with CH1-CL region of reduced mouse IgG2. Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.

Novel Biotinylated Phenylarsonous Acids as Bifunctional Reagents for Spatially Close Thiols: Studies on Reduced Antibodies and the Agonist Binding Site of ReducedTorpedoNicotinic Receptors

“…It has been known for over 20 years that gentle reduction of the interheavy chain disulfides in the hinge region yields an antibody that can strongly bind antigen but not C1q (28), the trigger for the complement cascade. Furthermore, electron microscopic (29), gel filtration (30), and immunological and X-ray scattering data (31) all show conformational changes in immunoglobulins upon reduction of the interheavy chain disulfide bonds. This site is a well-established target for antibody-labeling agents, and we infer that this is the most likely site of labeling by 3 since Fab fragments are not labeled (data not shown).…”