The use of liquid and gel isoelectric focusing as a means of further evaluating the degree of restriction of rabbit antibodies

Abstract

Antibodies from a homozygous rabbit hyperimmunized with type VIII pneumococcal vaccine was examined by isoelectric focusing in polyacrylamide gels. Significant quantities of electrophoretically restricted pneumococcal polysaccharide antibodies were obtained from single rabbit bleedings by preparative liquid isoelectric focusing of total immunoglobulins, obtained by salt precipitation, in a sucrose gradient using 1% w/v ampholytes covering 3 pH units. The method was modified to obtain the best resolution possible. The electrophoretic mobility of the electrofocused antibodies was compared by gel isoelectric focusing. The degree of restriction of the heavy and light polypeptide chains of the electrofocused antibody fractions was examined both by alkaline disc gel electrophoresis in urea and by isoelectric focusing in polyacrylamide gels in urea following complete reduction and alkylation of the focused antibody fraction. Isoelectric focusing in polyacrylamide gels in urea was found to be the best and easiest method to study the number and electrophoretic mobility of the fully reduced and alkylated light and heavy chains. Attempts were made to show that no molecular sieving occurred with the antibody heavy chains in 6.7% polyacrylamide gels. A number of electrofocused anti-pneumococcal polysaccharide antibody fractions yielded single light chain zones with different electrophoretic mobilities from a single antiserum suitable for comparative primary sequence analyses.