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Development of a CRF1 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System

oftware package GraphPad Prism v4.0. A Kd value of 0.4 nM (95% confidence intervals 0.3–0.5 nM) was estimated directly from the curve.

IC50 determination
Competitive binding of 0.3 nM [125I]Tyr0-CRF with urocortin (Fig 2A) and astressin (Fig 2B) was assessed and the IC50 value of each ligand calculated. Final concentrations in the wells were 0.039–160 nM and 0.005–500 nM respectively. Two separate batches of [125I]Tyr0-CRF were used for these evaluations, each with significantly different specific activities. This is demonstrated by the increase in specific IODs shown in Figure 2B. The IC50 value for urocortin was determined to be 3.5 nM (95% confidence interval range 2.7–4.5 nM) and the Ki value was 1.9 nM (95% confidence intervals 1.5–2.5 nM). The 50 value for astressin was 1.2 nM (95% confidence interval range 1.1–1.3 nM) and the Ki value was 0.6 nM (95% confidence intervals 0.6–0.7 nM).

Z’ analysis
Finally, a Z’ analysis was performed using 66 replicate values for “total” and NSB wells (4). The Z’ value indicated by Figure 3 is 0.64, which was well within the acceptable Z’ value range (0.5–1.0) and confirmed the robustness of the assay.


References
1. Steckler, T. and Holsboer, F. Biological Psychiatry 46, 1480–1508 (1999).

2. Hauger, R. L. et al. Pharmacological Review 55, 21–26 (2003).

3. Timpl, P. et al. Nature Genetics 19, 162–166 (1998).

4. Zhang, J. et al. Journal Biomolecular Screening 4 (2), 67–73 (1999).



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