Isolation and characterization of low and high affinity goat antibodies directed to single antigenic sites on human hemoglobin

A group of anti-hemoglobin antibodies from a goat immunized with human HbA₁ has been fractionated according to affinity. By elution of anti-HbA₁ antibodies bound to a β chain Sepharose column with a gradient of decreasing pH and increasing acetic acid concentration, the lower affinity antibodies eluted earlier from the column and were separated from the higher affinity antibodies. Fluorescence quenching titrations with HbA₁ and the β chains showed the first and the last of these fractions to be directed toward single antigenic sites on the β chain. Comparisons of their interactions with several hemoglobin variants establish that they bind to different sites on the β chain. The antigenic site on the β-chain to which the high affinity fraction binds involves the β⁶ position since its affinity for HbS (β⁶ glu → val) and Hb Leiden (β^{6 or7} deleted) is significantly lower than its affinity for HbA₁.