Amino acid substitutions outside a preselected antigenic region in hemoglobin affect the binding to monoclonal antibodies obtained by immunization with the synthetic region

It is often assumed that amino acid substitutions outside a protein antigenic site have no effect on the reactivity of a protein variant with antibodies, especially monoclonal antibodies (mAbs). Substitutions that exert an effect on the reactivity of a protein variant with mAbs are frequently considered to be within the antigenic site of the mAb. To test this assumption, two mAbs IgG_l(k) and IgG_2a (k)] were prepared by immunization with a synthetic peptide corresponding to region 63–78 of the chain of human hemoglobin (Hb). The peptide was used as an immunogen in its free form (i.e., without conjugation to a carrier), so that the results will not be made ambiguous by peptide modification nor by an immune response to sites spanning peptide and protein carrier. In addition to their reaction with human Hb, the mAbs were also studied with four primate Hbs which had no substitutions within region 63–78 and only a limited number of substitutions which occurred outside of, and at considerable distances in three-dimensional (3D) structure from, this region. Inhibition studies revealed substantial differences in the binding affinities of some of the primate Hbs, relative to human Hb. Some of the substitutions caused major decreases in binding, although they were at considerable distances in the 3D structure from the indicated site residues. It is concluded that substitutions in a protein, even when distant from an antigenic site, can exert major influences on the protein's reactivity with anti-site mAbs.