Properties of goat anti-human hemoglobin antibodies fractionated on subunit affinity columns

The antibodies to human hemoglobin A, (HbA₁) in the sera of two goats were fractionated on the basis of their ability or inability to bind to the isolated α and β subunits of the hemoglobin. The fractionation was carried out by a series of affinity chromatography steps and the characterization of the binding of the antibody populations thus obtained to HbA₁, α and β revealed some predominating characteristics common to the two goat sera. In roughly proportional yields, the anti-HbA₁ in the sera were fractionated into three major catergories: antibodies which bind only to HbA₁ and β chains, antibodies which bind only to HbA₁ and α chains and antibodies which bind to HbA₁ and both α and β chains. One goat also had an antibody population that would bind HbA₁ but neither α nor β. There was, in both sera, a larger proportion of α binding control to β binding antibodies and of α-ß non-cross-reacting compared to α-ß cross-reacting antibodies. Upon consideration of the amino acid sequence and three-dimensional structures of both the hemoglobin antigen and the host's own hemoglobin with respect to sequence differences and the exposure and clustering of such differences, the existence and proportions of the diffrent antibody populations can be explained.