The distribution of antibodies for erythrocytes in chromatographic fractions of early and late immune sera.

The sera of rabbits injected intravenously with sheep, human O and beef erythrocytes respectively were collected at various stages during immunization and were fractionated by elution from anion exchange DEAE cellulose columns with phosphate solutions of increasing ionic strength and decreasing pH. In early sera prepared against all three types of cells the predominant agglutinating activity was associated with fractions containing macroglobulins eluted by phosphate buffers of higher molarity. On continued immunization, however, agglutinins appeared in fractions eluted from the cellulose by phosphate solutions of lower molarity. The latter agglutinins sedimented more slowly on ultracentrifugation than the former. The early antibodies showed a greater ratio of haemolytic agglutinating activity for sheep and beef cells than those appearing later. The haemolytic activity of human O cell antibodies was relatively weak but these antibodies showed a change in chromatographic distribution during immunization similar to that of antibodies for sheep and beef cells. The failure of early agglutinins to react with rheumatoid factor was shown to be due to the fact that the agglutinins appearing first are macroglobulins which are not reactive.