[48] Procedures for binding an antibody to receptor-bound interferon

Publisher Summary This chapter describes procedures for binding an antibody to receptor-bound interferon. Antibodies recognizing receptor-bound interferons (IFNs) are interesting for various reasons. They may be used to isolate IFN/receptor complexes, to analyze structure/function relationships of IFNs, to analyze the fate of IFNs bound to the surface of living cells, or to identify individual cells with, particularly high or particularly low numbers of IFN receptors. Ideally, such antibodies should bind to receptor-bound IFN with high affinity, they should not displace IFN from its receptor, they should not interfere with IFN binding to receptors, and, consequently, not neutralize IFN activity. Antibodies specific for one epitope (or a narrow range of epitopes) of IFNs may satisfy at least some of these criteria. Although the procedure for binding an antibody to the carboxyl terminus of receptor-bound IFN appears straightforward, there is no guarantee that other monoclonal or polyclonal antibodies recognizing the ultimate carboxyl terminus of IFN are able to recognize receptor-bound IFN. IFN undergoes several conformational changes after binding to its specific receptor, and IFN with such conformational changes is recognized only by particular antibodies. Antibody III/21 may be unique in that it binds stoichiometrically to IFN bound to the surface of Mardin–Darby Bovine Kidney (MDBK) cells. It binds to both that portion of the IFN that is bound specifically to receptors as well as that portion that is bound nonspecifically to the cells.