Quantitative Studies on the Bis Diazotized Benzidine Method of Hemagglutination

Abstract

Quantitative studies on the BDB method of hemagglutination revealed that an average of 5 × 10⁵ molecules of C¹⁴-anti-BSA rabbit globulin were fixed to each BSA sensitized rabbit erythrocyte which was agglutinated in antibody excess. Similar results were obtained in experiments using different suspensions of erythrocytes and freshly prepared BDB. There was a strictly linear relationship between the number of sensitized cells added and the number agglutinated in antibody excess. As the number of sensitized cells added per tube increased 6-fold, the quantity of homologous antibody bound per cell decreased by 50%. When the agglutinated red blood cells were hemolyzed with water, all of the bound C¹⁴ antibody was found in the stroma. Using H³-labeled BSA, it was demonstrated that an average of 1.3 × 10⁶ molecules of antigen were coupled to each erythrocyte during the BDB sensitization procedure. The significance of these findings has been discussed.