Interactions between Fab and Fc regions in liganded immunoglobulin G: A study employing the antigenic polymorphism and multivalency of the human red-cell membrane

Previous studies indicated that the inter-y chain disulphide bonds restrict segmental flexibility within the Fc region of human IgG; the present study suggests that there are also non-covalent hinge-region restrictions of flexibility in the liganded molecule. Staphylococcus aureas protein A-Sepharose-passed F(ab′)₂ fragments from IgG fractions of blood-group antisera were tested in parallel with the parent antibodies in haemagglutination tests. At molar-equivalence in protein, the titre of F(ab′)₂ from incomplete antibodies was 2- to 3-fold greater than the titre of the intact antibody (p < 0.001). Increased activity occurred whether enzyme or albumin techniques were used to convert the incomplete antibodies to agglutinins, and was particularly striking with Kell blood-group antibodies where F(ab′)₂ fragments were weak saline agglutinins. Antiglobulin tests, using anti-κ, revealed small differences in intrinsic antigenbinding activity between IgG and F(ab′)₂ fractions, but these differences were either of inverse relationship or insufficient to account for the increased agglutinating activity of F(ab′)₂ compared with the whole molecule. By contrast, with IgG saline agglutinins [anti-κ (anti-glycophorin A^M) and anti-B], the apparent freedom introduced by peptic removal of Fc, or by reduction of interchain disulphide bonds, was inimical to agglutination, as would be predicted from other studies (Hornick & Karush, 1972; Crothers & Metzger, 1972). These results show that the Fe region imposes limitations on full segmental flexibility of Fab regions in liganded IgG.A comparison of the intrinsic and functional antigen-binding activities of IgG anti-M and anti-B confirmed that both antibodies engage in substantial monogamous bivalency. That the relative monogamous bivalency of F(ab′)₂ and IgG anti-M was unaffected by an increased mean separation of M sites on the cell surface suggests that it is predominantly dimers of glycophorin a^M, in situ, which generate the monogamous bivalency and that, on M/N red cells, some glycophorin-A^M molecules are associated as homologous dimers. For IgG anti-A and anti-B, the known decreased affinity with A₂ and A₁B red cells can be explained by the fewer opportunities for monogamous bivalency. Using the increased flexibility in F(ab′)₂ and reduced-alkylated IgG anti-D as molecular probes for spatial relationships between D antigens, (i) a marked deviation from an average separation of antigens was found on normal red cells under conditions of agglutination, and (ii) an early effect of trypsin-treatment (< 2 min) which permits limited redistribution of D sites was detected.Mildly reduced IgG3 exhibited significantly greater flexibility than reduced IgG1, presumably reflecting the extended γ3 hinge region. Unreduced IgG3, however, did not show the increased Fab-region flexibility shown by F(ab′)₂ fragments on the IgG1 sub-class. In reduced IgG molecules, Cγ2 and Cγ3 domains retained much of their native surface topography.