A rapid and sensitive binding assay for growth hormone releasing factor.

Abstract

A binding assay for growth hormone releasing factor (GRF) has been developed using scintillation proximity assay (SPA) technology. Binding conditions were validated by several criteria. Equilibrium binding was attained within three hours at 22 degrees C in crude membrane fractions of HEK293 (293-P2) and GH4C1 (GH4-P1) cells transfected with the porcine GRF receptor. Saturation binding isotherms produced a KD of 296 pM and a Bmax of 4.7 pmols/mg membrane protein in 293-P2 cells. Cells not expressing the GRF receptor displayed no specific binding for the ligand. Competition binding curves produced the following rank order of potency for tested peptides: GRF analogs D-Ala2 = D-Arg2 (IC50 approximately 1 nM) >> PACAP > secretin, VIP (EC50 > 100 nM). Somatostatin (SRIF) binding was also adapted to the SPA format in a GH4C1 cell line transfected with the SRIF receptor subtype 2 (SSTR2) and in HEK293 cells transfected with the SRIF receptor subtype 5 (SSTR5). This assay represents a major improvement for binding measurements of these and potentially many other ligands for G-protein linked receptors, requiring no separation of bound from free hormone, allowing detailed pharmacological evaluations and enabling measurement of equilibrium binding in real time. In the 96-well format, it is suitable for high throughput screening.