INTERACTIONS BETWEEN FAB AND FC REGIONS IN LIGANDED IMMUNOGLOBULIN-G - 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 AM) 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 aM, in situ, which generate the monogamous bivalency and that, on M/N red cells, some glycophorin-AM 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. © 1979.