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Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System

range 5.3–9.4 nM) and the mean Ki value was 2.7 nM (95% confidence intervals 2.1–3.6 nM). Figures 3A and 3B are representative of the data obtained.

Time course
A time course was performed using standard reagent concentrations detailed in the protocol with an increased incubation time of 18 h. The assay appeared to be stable for at least 18 h as demonstrated by the curve in Figure 4.

Z’ analysis
Finally, two independent Z’ analysis (5) evaluations were performed, each using 48 replicate values for "total" and NSB wells. DMSO was present at a final concentration of 2% (v/v) in each well. A mean Z’ value of 0.82 was generated. Figure 5 is representative of the data obtained. This is well within the acceptable Z’ value range (0.5–1.0) and confirmed the robustness of the assay.


Conclusion
The histamine H1 receptor binding assay has been successfully miniaturized to a 384-well format using the LEADseeker Multimodality Imaging System and is suitable for adaptation to automated screening formats. The assay is tolerant to DMSO up to a concentration of 2% (v/v), robust; achieving a Z’ value of 0.82 and a stability window of at least 18 h.


References
1. Hill, S. J. et al. Pharmacological Reviews 49, 253–278 (1997).
2. Oda, T. et al. Journal Biological Chemistry 275, 36781-36786 (2000).
3. Curran, M. P. et al. Drugs 64, 523-561 (2004).
4. Matsubara, M. et al. Biochemical Pharmacology 69, 433-449 (2004).
5.Zhang, J. et al. Journal Biomolecular Screening 4 (2), 67–73 (1999).
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