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The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System

(0.12 pM–1.172 µM), adrenomedullin (3.1 pM–30.4 µM), amylin (2.1 nM–32.8 µM), and CGRP (8–37) (3.1 pM–30 µM).

For CGRP, the IC50 value was 0.26 nM (95% confidence interval range 0.2 to 0.33 nM) and the Ki value was 0.16nM (95% confidence interval range 0.12 to 0.22 nM). For adrenomedullin, the IC50 value was 17.2 nM (95% confidence interval range 10.8 to 27.5 nM) and the Ki value was 10.5 nM (95% confidence interval range 6.6 to 16.8 nM). For amylin, the IC50 value was 563 nM (95% confidence interval range 371 to 856 nM) and the Ki value was 343 nM (95% confidence interval range 226.3 to 521.6 nM). For CGRP (837), the IC50 value was 3.86 nM (95% confidence interval range 2.7 to 5.4 nM) and the Ki value was 2.35 nM (95% confidence interval range 1.7 to 3.3 nM).

Time course
A time course experiment was carried out using reagent concentrations as described in the protocol. Measurements were made over an assay incubation time of 18 h. The assay reached equilibrium after 4 h incubation at room temperature, and the signal was stable for at least 14 h (Fig 4).

Z’ analysis
A Z’ analysis was carried out using 48 replicate values for total and NSB wells. Z’ was determined to be 0.64 (Fig 5), which confirmed the robustness of the assay (7).


Conclusions
The CGRP receptor-binding assay has been successfully miniaturized to a 384-well format on LEADseeker Multimodality Imaging System. The assay is suitable for high-throughput screening, and is tolerant up to 1% DMSO. The assay is robust exhibiting a Z’ value of 0.64 and it has a stable signal of at least 14 h.
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