( p Competition Binding Assays ...)Development of Radioligand Binding Assays for the Motilin Receptor Using ScreenReady Targets.

p>Competition Binding Assays
The affinity of unlabeled motilin for the motilin receptor was then determined in competition assays. A competition assay of [¹²⁵I]-motilin binding by motilin on motilin ScreenReady Targets showed that a monophasic competition curve was obtained with a K_i value of 5.4 nM (Figure 3). This value was in good agreement with reported values (Thielemans et al ., 2001). Taken together, results from saturation and competition experiments clearly show that genuine pharmacological properties (K_d, K_i) can be obtained for motilin ScreenReady Targets in microformatted FlashPlate assays.

Intra-plate Variability
Intra-plate variability of [¹²⁵I]-motilin binding using motilin ScreenReady Targets was assessed. Membranes from CHO cells expressing the human motilin MTL-R1A receptor were immobilized into 96-well FlashPlate microplates and binding assays were performed using 48 wells for total binding determination and 48 wells for non-specific binding determination (Figure 4). The CV and S/B values led to a calculated Z factor (Zhang et al ., 1999) of 0.75, showing the robustness of the assay.

Conclusion

Using PerkinElmers proprietary coating procedure, we have developed ScreenReady Targets for homogeneous radioligand binding assays. The results obtained using the human motilin MTL-R1A receptor show that genuine pharmacological properties (K_d, K_i) can be determined and correspond to reported values (Feighner et al ., 1999; Thielemans et al ., 2001). Pharmacological analysis of the coated receptors showed that they maintain their binding properties, even after prolonged storage (3 months) at 4C. Low intra-plate variability (3.5%) on [¹²⁵I]-motilin binding and a robust Z factor (0.75) demonstrate the usefulness of
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