A novel method is described for the quantitative study of haemagglutination in terms of the continuous and direct sizing and counting of aggregates of red blood cells (RBC) as they settle in free suspension. This sedimentation-enumeration (SE) method was used to estimate the concentration of haemagglutinating particles in terms of the number of new RBC-RBC bonds formed. In haemagglutination by Semliki Forest virus (SFV), the formation of RBC-RBC bonds is interpreted in terms of two competitive and near first-order reactions: the rate of inactivation of SFV haemagglutinin at low values of pH, and the rate of adsorption of residual SFV haemagglutinin by RBC. The SE method provides an estimation of the concentration of virus in terms of haemagglutinating activity which is independent of container wall effects and of the concentration of RBC. Results were compared with parallel estimates of the concentrations of infective and physical particles. At the optimum pH 6.3 for haemagglutination, and at a concentration of 10 RBC/ml., about 7 particles of haemagglutinin were required for the formation of one RBC-RBC bond.

At low concentrations of SFV haemagglutinin, the distribution of single RBC and of aggregates of RBC was consistent with a statistical-mechanical theory of aggregation which provides a basis for the interpretation of the mechanism of haemagglutination. The distributions observed in this study were not consistent with the ‘dimers-only’ hypothesis of Levine, Puck & Sagik (1953) which was used by Cheng (1961) in an early study of haemagglutination by SFV.


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