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Journal of Biomolecular Screening
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Validation of a High Throughput Scintillation Proximity Assay for 5-HydroxytryptaminelE Receptor Binding Activity

Steven D. Kahl

Technology Core Research, Lilly Research Laboratories, Indianapolis, Indiana 46285

Frederick R. Hubbard

Sphinx Pharmaceuticals, A Division of Eli Lilly and Company, Durham, North Carolina 27707

G. Sitta Sittampalam

Technology Core Research, Lilly Research Laboratories, Indianapolis, Indiana 46285

Joseph M. Zock

Natural Products Research and Development, Lilly Research Laboratories, Indianapolis, Indiana 46285

Membranes prepared from stable transfected cells expressing the human gene encoding a functional 5-hydroxytryptaminelE (5HT1E) receptor were used to investigate high-affinity [3H]serotonin ([3H]5-HT) binding with scintillation proximity assay (SPA) technology. In this nonseparation format, membranes are captured by WGA-coated SPA fluoromicrospheres for detection of receptor-bound radioligand. Total binding observed was approximately 2000 cpm compared to a nonspecific signal of 100 cpm determined in the presence of 10,uM unlabeled 5-HT. Non-proximity effects (NPE) for the radiolabel and SPA beads averaged less than 100 cpm. Saturation binding analysis yielded an equilibrium dissociation constant (Kd) of 5.38 ± 0.43 nM and a maximum binding capacity (Bmax) of 6.42 + 0.15 pmol/mg protein. Competition with unlabeled serotonergic compounds demonstrated a specificity of the assay with rank potencies (5-HT > a-Me-5-HT > 2-Me-5-HT > 5-CT) similar to those observed using traditional fitration techniques. The variability of the assay and the stability of all reagents were investigated to validate the assay for extended use throughout a typical high throughput screen operation lasting several months.

Journal of Biomolecular Screening, Vol. 2, No. 1, 33-40 (1997)
DOI: 10.1177/108705719700200107
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