Characterization of [125I-Tyr0]-corticotropin releasing factor (CRF) binding to the CRF binding protein using a scintillation proximity assay

We describe the characterization of high affinity [I-125-Tyr(0)]-human CRF binding to purified recombinant human CRF-binding protein (CRF-BP) using a scintillation proximity assay (SPA). For this stable nonseparation technique developed in 96 well microtiter plates, biotinylated CRF-BP is captured by streptavidin-coated SPA beads for the detection of bound [I-125-Tyr(0)]-CRF. Unbound [I-125-Tyr(0)]-CRF represented little or no signal in the assay. Total binding observed was greater than 5000 cpm with a nonspecific signal of < 100 cpm determined in the presence of excess unlabeled human CRF. A comparison of the SPA method with a charcoal precipitation method confirmed that the biotinylation procedure did not adversely affect affinity of the CRF-BP for [I-125-Tyr(0)]-CRF. Saturation binding analysis yielded an apparent equilibrium dissociation constant (K-d) of 208 +/- 5.0 pM (+/- S.D., n = 3). An inhibition constant (K-i) for unlabeled CRF was calculated to be 0.22 +/- 0.03 nM (+/- S.D., n = 8) and a pharmacological profile for eight CRF-related neuropeptides gave a rank potency similar to previously reported results. Finally, the assay variability was assessed with intra- and inter-plate coefficients of variation which were less than 5% each. (C) 1998 Elsevier Science B.V. All rights reserved.