Figure 6 Differentiation of information provided by different fluorimetric readout parameters.

one antibody. A one-dimensional brightness analysis allows for a differentiation between antibodies carrying either one or two ligands but not between free ligand and a single ligand bound by an antibody.

Using EVOTEC's multidimensional fluorescence analysis, whereby thepo-larization ratio and the brightnesses of the particles are determined in parallel, each individual step of such a multiple binding reaction can be analyzed. The results of these studies are shown in Figure 7, where the individual components of a multicomponent system can be resolved in a single measurement. Here it can be seen that the concentration of free ligand decreases with increasing antibody concentration. The population of antibodies bearing two ligand molecules reaches a maximum at an antibody dilution of 2 X 10~5 and begins to decrease as antibody becomes limiting. At this point the number of antibodies bearing a single ligand

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