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

uasi-coincident averaging and 3 × 3 binning.


Results
Kd determination

Saturation binding was performed with dilutions of [pyridinyl-5-3H]Pyrilamine to give a range of concentrations from 0.13 to 33.2 nM in the wells. Plates were incubated for 5 h at room temperature and in darkness prior to imaging. Figure 1 shows the saturation curve, which was fitted using non-linear regression with the data analysis software package GraphPad Prism v4.0. Over two experiments the mean Kd value was determined to be 3.7 nM (95% confidence intervals 3.1–4.4 nM), estimated directly from the curve. Figure 1 is representative of the data obtained.


DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.25% (v/v) to 5% (v/v) in the wells. The results shown in Figure 2 indicate the assay was tolerant to a final concentration of 2% (v/v). However, the signal was only slightly reduced in the presence of 5% DMSO.

IC50 determination
Competitive binding of 5.6-nM [pyridinyl-5-3H]Pyrilamine with pyrilamine (Fig 3A) and triprolidine (Fig 3B) was assessed and the IC50 of each ligand calculated. Final concentrations in the well ranged from 0.005 to 10 000 nM respectively. DMSO was present at a final concentration of 2% (v/v) in each well.

Over two experiments, the mean pyrilamine IC50 value was determined to be 14.6 nM (95% confidence interval range 10.6–20.7 nM) and the mean Ki value was 5.7 nM (95% confidence intervals 4.2–7.9 nM). The mean triprolidine IC50 value was determined to be 6.9 nM (95% confidence interval
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