title: Using nonfluorescent Förster resonance energy transfer acceptors in protein binding studies.
abstract: The purpose of this article is to highlight the versatility of nonfluorescent Förster resonance energy transfer (FRET) acceptors in determination of protein equilibrium dissociation constants and kinetic rates. Using a nonfluorescent acceptor eliminates the necessity to spectrally isolate the donor fluorescence when performing binding titrations covering a broad range of reagent concentrations. Moreover, random distribution of the donor and acceptor chromophores on the surface of proteins increases the probability of FRET occurring on their interaction. Three high-affinity antibodies are presented in this study as characteristic protein systems. Monoclonal antibody (mAb) 106.3 binds brain natriuretic peptide (BNP)5-13(C10A) and full-length BNP1-32 with the dissociation constants 0.26+/-0.01 and 0.05+/-0.02 nM, respectively, which was confirmed by kinetic measurements. For anti-hCG (human chorionic gonadotropin) mAb 8F11, studied at two incorporation ratios (IRs=1.9 and 3.8) of the nonfluorescent FRET acceptor, K(D) values of 0.04+/-0.02 and 0.059(-0.004)(+0.006) nM, respectively, were obtained. Likewise, the binding of goat anti-hamster immunoglobulin G (IgG) antibody was not affected by conjugation and yielded K(D) values of 1.26+/-0.04, 1.25+/-0.05, and 1.14+/-0.04 nM at IRs of 1.7, 4.7, and 8.1, respectively. We conclude that this FRET-based method offers high sensitivity, practical simplicity, and versatility in protein binding studies.
authors: Diagnostic Research, Abbott Diagnostics Division, Abbott Park, IL 60064, USA.
authors: Qiaoqiao
authors: Q
authors: Ruan
authors: Joseph P
authors: JP
authors: Skinner
authors: Sergey Y
authors: SY
authors: Tetin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D002264:Carboxylic Acids / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000276:immunology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D020097:Natriuretic Peptide, Brain / Q000276:immunology; D011485:Protein Binding; D013388:Succinimides / Q000378:metabolism; D017186:Titrimetry
chemical_list: C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; D000911:Antibodies, Monoclonal; D002264:Carboxylic Acids; D006063:Chorionic Gonadotropin; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D008024:Ligands; D013388:Succinimides; D020097:Natriuretic Peptide, Brain