Characterization of model antigens composed of biotinylated haptens bound to avidin

Model antigens of defined structure and limited valency have been prepared by binding 2,4-dinitrophenyl (DNP)-biotin haptens of different lengths to the biotin binding sites of avidin. These complexes have been characterized structurally by spectroscopic methods and functionally by testing their ability to crosslink cell-bound anti-DNP immunoglobulin E-receptor complexes to stimulate degranulation of rat basophilic leukemia cells. One of these haptens, 1-DNP-amino-12-biotinamido dodecane, is shown to have only two tight binding sites per avidin molecule, and the resulting bivalent (DNP-biotin)-avidin antigen is found to stimulate substantial degranulation. Another DNP-biotin hapten that is approximately 10 A longer has four tight binding sites per avidin and, when bound to avidin, has greater activity similar to a highly DNP-conjugated multivalent antigen. These and other (DNP-biotin)-avidin complexes described here represent a new group of chemically well-defined antigens that are useful for studying the binding to and functional crosslinking of cell-bound immunoglobulins in immunological responses.