Abstract
The D-optimal design, a minimal sample design that minimizes the volume of the joint confidence region for the parameters, was used to evaluate binding parameters in a saturation curve with a view to reducing the number of experimental points without loosing accuracy in binding parameter estimates. Binding saturation experiments were performed in rat brain crude membrane preparations with the opioid μ-selective ligand [ 3H]-[D-Ala 2,MePhe 4, Gly-ol 5]enkephalin (DAGO), using a sequential procedure. The first experiment consisted of a wide-range saturation curve, which confirmed that [ 3H]-DAGO binds only one class of specific sites and non-specific sites, and gave information on the experimental range and a first estimate of binding affinity (K a), capacity (B max) and non-specific constant (k). On this basis the D-optimal design was computed and sequential experiments were performed each covering a wide-range traditional saturation curve, the D-optimal design and a splitting of the D-optimal design with the addition of 2 points(± 15% of the central point). No appreciable differences were obtained with these designs in parameter estimates and their accuracy. Thus sequential experiments based on D-optimal design seem a valid method for accurate determination of binding parameters, using far fewer points with no loss in parameter estimation accuracy.
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