Isolation and Characterization of Electrophoretically Homogeneous Rabbit Antihapten Antibody Populations

The structural heterogeneity of light polypeptide chains derived from rabbit antibodies directed against p-azophenyltrimethylammonium (Ap) and p-azobenzenearsonate (Rp) haptens from individual heterozygous rabbits was investigated. Antibodies were purified by adsorption on specific solid immunoadsorbents, followed by preparative liquid isoelectric focusing at either pH 5 to 8 or pH 7 to 10. The focused anti-Ap antibody fractions were refocused in pH gradients extending only 0.5 pH unit on either side of their respective isoelectric points. A number of focused antibody fractions demonstrated electrophoretic, structural, and functional features characteristic of homogeneous antibody populations. Amino-terminal sequences of the kappa light chains, obtained by mild reduction and alkylation followed by gel filtration, were determined for nine focused fractions of anti-Ap antibody from one rabbit, seven focused fractions of anti-Rp antibody from a second rabbit, and two focused fractions of anti-Rp antibody from a third rabbit. Amino acid sequence analyses extended for 6 to 39 positions (a few included the first hypervariable region) depending upon the quantity of light chain obtained from the focused fractions. Sequences obtained from light chains isolated from serum prior to immunization immunoglobulins or from specifically purified unfractionated antibodies were heterogeneous (2 to 4 residues at most positions), while those from the major focused antibody fractions were as homogeneous, for most of the first 25 to 30 positions, as human kappa Bence-Jones proteins sequenced by the same methods. The light chains from the focused antibodies demonstrated significant microheterogeneity and nonidentity within limited areas (positions 31 to 34). Focused antibody light chains had sequences representative of either the Vkii or Vkiii subgroups. This approach, therefore, permits structural studies to be performed on the homogeneous antibody constitutents of heterogeneous antibody preparations and demonstrates that the heterogeneity is relatively restricted to limited areas.