MONOCLONAL-ANTIBODIES TO HUMAN SPLEEN FERRITIN .2. LOCALIZATION OF EPITOPES AND QUANTITATIVE PARAMETERS OF ANTIGEN-BINDING

Interactions of three monoclonal antibodies with human spleen ferritin were investigated. The contents of active antigen-specific antibodies in ascites fluid (22-37%), total IgG fraction (35-50%), and affinity eluate of the antibody HSF102 (88%) were determined by titration with immobilized antigen. A mathematical model developed by the authors and [I-125]ferritin binding assays showed that the affinity constants for the monoclonal antibodies range from 6.10(8) to 3.10(9) M-1. Each of the three antibodies strongly (by 77-95%) inhibits binding of rabbit p olyclonal antibodies to ferritin, indicating compact clustering of the dominant immune epitopes and the close neighborhood of binding sites for mono- and polyclonal antibodies. Competitive binding assays and ad ditivity tests with the monoclonal antibodies showed that the epitope specific for HSF102 antibody is localized in the periphery of the clus ter and located remote from the epitopes specific for HSF101 and HSF10 3 antibodies by a distance that allows for noncompetitive binding of t he two antibodies. Competition between HSF101 and HSF103 indicates tha t their epitopes overlap. A maximum of four [I-125]IgG molecules can b ind simultaneously to a ferritin molecule. This number characterizes t he maximum valency of the antigen in reactions with IgG-class antibodi es.