Case Study of Multidimensional FCS Readout

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A novel assay based on a multiparameter fluorescent readout is described below. A theophylline/antitheophylline antibody interaction was chosen as a pharmacologically relevant assay system in order to demonstrate the full potential of EVOTEC's multidimensional fluorescence analysis based on its proprietary FCS+ detection platform.

Theophylline therapy has been a cornerstone of asthma therapy through the years. Theophylline inhibits the breakdown enzyme phosphodiesterase with a resultant increase in cAMP concentrations, which results in smooth muscle cell relaxation of the bronchial tree. In view of the very small margin between therapeutic effects and toxicity, individualization of dose is mandatory with theophylline therapy. Therefore a strong demand for highly sensitive assays and detection technology exists in order to fine tune the theophylline level in serum [9-11].

The antibody used for this study was a polyclonal antitheophylline serum. The most interesting antigens were chosen to be those bearing TAMRA labels. Reference Kds for antibody and ligand titration series were determined by FCS and compared with those obtained by the multidimensional fluorescence analysis (Fig. 5).

The mode of interaction is depicted in Figure 6. For a precise description of this antigen-antibody interaction, a model was chosen assuming two identical and independent binding sites. L represents the ligand and R the antibody with two binding sites. Three reaction diagrams show the reaction participants, which can be discriminated from one another by two-dimensional analysis, FCS, and one-dimensional brightness analysis.

Using FCS, one is able to discriminate between the bound and the non-bound fraction of the ligand based on the translational diffusion properties of the molecules but not between the state where either one or two ligands are bound by

Figure 5 FCS measurement of theophylline and antitheophylline interaction. The preliminary FCS studies indicated well-defined binding characteristics of the ligand and reinforced the suitability of the system for a homogeneous assay. The data were fitted according to a single binding site model.

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