Evaluation of electrochemiluminescent technology for inhibitors of granulocyte colony-stimulating factor receptor binding.
An electrochemiluminescent (ECL) assay was developed to identify compounds that inhibit the interaction of granulocyte colony-stimulating factor (GCSF) with its recombinant human receptor. The ECL technology uses a tris-(bipyridine) chelate of ruthenium, which, in the presence of excess tripropylamine, undergoes a redox reaction cycle to produce light. Paramagnetic beads with primary antibody were coated with secondary anti-GCSF receptor antibody, which were then bound with GCSF receptor. These samples were incubated with ruthenylated GCSF in the presence and absence of test compounds. The bead density, receptor and ligand concentrations, and incubation time were optimized in the assay. A set of mixed compound plates was screened to examine the feasibility of using this technology in high throughput screening. The results from this format were found to be comparable to the assay performed using a time-resolved fluorescence format.
Gopalakrishnan
S M
SM
Abbott Laboratories, Abbott Park, IL 60064, USA.
Warrior
U
U
Burns
D
D
Groebe
D R
DR
D004396:Coloring Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D016179:Granulocyte Colony-Stimulating Factor / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D008163:Luminescent Measurements; D008863:Microspheres; D010084:Oxidation-Reduction; D011437:Propylamines / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017975:Ruthenium Compounds / Q000378:metabolism
D004396:Coloring Agents; D011437:Propylamines; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D017975:Ruthenium Compounds; C096226:tripropylamine; D016179:Granulocyte Colony-Stimulating Factor; D004121:Dimethyl Sulfoxide