Studies of hyperimmune restricted and partially restricted anti‐pneumococcal polysaccharide antibodies from allotype‐defined pedigreed rabbits. I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques

Four heterozygous rabbits (a2 a2, b4 b5; a1 a2, b6 b9; a2 a2, b5 b9; a3 a3, b4 b5) and one homozygous rabbit (a2 a2, b6 b6) were hyperimmunized with either type 3 or 8 pneumococcal vaccines. Four antisera were partially restricted and one antiserum was highly restricted. The immunoglobulins (Ig) were obtained by salt precipitation, ion‐exchange chromatography, and affinity chromatography. The total Ig, obtained by salt precipitation, were fractionated by preparative liquid isoelectric focusing (IEF) at various pH ranges. From each antiserum a number of fractions were obtained with significantly different isoelectric points. Many major fractions had reduced electrophoretic heterogeneity when compared with the Ig isolated before IEF. These fractions were tested for isoelectric purity by analytical IEF in polyacrylamide gels and by preparative refocusing over a narrow pH range. After complete reduction and alkylation, the light and heavy polypeptide chains of the focused antibody fractions were examined both by disc electrophoresis in alkaline urea gels and by IEF in polyacrylamide gels containing urea. From each antiserum, a number of fractions were obtained which yielded single light chains often with different electrophoretic mobilities. This method provided a successful means of preparatively obtaining different antibody fractions from a single antiserum.