Characterization of [ 125I-Tyr 0]-corticotropin releasing factor (CRF) binding to the CRF binding protein using a scintillation proximity assay

• Published in: Journal of neuroscience methods 1998-09, Vol.83 (2), p.103-111
• Main Authors: Kahl, S.D, Liu, X.-J, Ling, N, De Souza, E.B, Gehlert, D.R
• Format: Article
• Language: English
• Subjects: [ 125I-Tyr 0]-CRF binding; Animals; Binding, Competitive; Biological and medical sciences; Biotin; Charcoal; CHO Cells; Chromatography, Affinity - methods; Corticotropin releasing factor; Corticotropin-Releasing Hormone - analysis; Corticotropin-Releasing Hormone - metabolism; CRF-binding protein; Cricetinae; Fundamental and applied biological sciences. Psychology; General aspects. Models. Methods; Humans; Iodine Radioisotopes; Microspheres; Rats; Recombinant Proteins - analysis; Recombinant Proteins - metabolism; Scintillation Counting - methods; Scintillation proximity assay; Tyrosine; Vertebrates: nervous system and sense organs
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/S0165-0270(98)00059-4

We describe the characterization of high affinity [ 125I-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 [ 125I-Tyr 0]-CRF. Unbound [ 125I-Tyr 0]-CRF represented little or no signal in the assay. Total binding observed was greater than 5000 cpm with a nonspecific signal of