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 [125I-Tyr0]-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 [125I-Tyr0]-CRF. Unbound [125I-Tyr0]-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 [125I-Tyr0]-CRF. Saturation binding analysis yielded an apparent equilibrium dissociation constant (Kd) of 208 +/- 5.0 pM (+/- S.D., n = 3). An inhibition constant (Ki) 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.
Kahl
S D
SD
Research Technologies and Proteins, Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285, USA. skahl@lilly.com
Liu
X J
XJ
Ling
N
N
De Souza
E B
EB
Gehlert
D R
DR
D000818:Animals; D001667:Binding, Competitive; D001710:Biotin; D016466:CHO Cells; D002606:Charcoal; D002846:Chromatography, Affinity / Q000379:methods*; D003346:Corticotropin-Releasing Hormone / Q000032:analysis* / Q000378:metabolism*; D006224:Cricetinae; D006801:Humans; D007457:Iodine Radioisotopes; D008863:Microspheres; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012588:Scintillation Counting / Q000379:methods; D014443:Tyrosine
D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D002606:Charcoal; D014443:Tyrosine; D001710:Biotin; D003346:Corticotropin-Releasing Hormone