PERTURBATION OF ANTIBODY BOUND BIFLUORESCENT-LIGAND PROBE BY POLYCLONAL ANTI-METATYPE ANTIBODIES INTERACTING WITH EPITOPES PROXIMAL TO THE LIGANDED ANTIBODY ACTIVE-SITE

General localization of metatypic determinants recognized by polyclona l anti-metatype antibodies relative to the antibody active site of the high-affinity anti-fluorescein monoclonal antibody 4-4-20 was achieve d through use of a unique bifluorescent-ligand probe. The fluorescent probe possessed intrinsic energy-transfer properties with the fluoresc ein hapten serving as the energy acceptor. The donor group 5-(2-iodoac etyl) aminoethylaminonaphthalene-1-sulfonic acid (IAE DANS) proved env ironmentally sensitive both to binding of the FITC-cys-AEDANS ligand a nd to subsequent anti-metatype antibody interactions involving the ant ibody variable domains of 4-4-20. Spectral changes in ligand-conjugate d AEDANS upon specific reactivity of the antibody with FITC suggested secondary interactions between AEDANS and the topological protein surf ace adjacent to the 4-4-20 active site. Results indicated that some an ti-metatype antibodies (Fab fragments) within the polyclonal populatio n bound to sites immediately surrounding the liganded active site and perturbed the interactions of AEDANS with topological sites. The resul ts are discussed in terms of the types of interactions that may occur between the AEDANS moiety and the 4-4-20 antibody protein surface and subsequent perturbation of those interactions by anti-metatype antibod ies.