title: A fast capillary electrophoresis method to assess the binding affinity of recombinant antithrombin toward heparin directly from cell culture supernatants.
abstract: With the aim to determine the binding affinity of a new generation of recombinant antithrombin (AT) toward heparin, we developed a dynamic equilibrium-affinity capillary electrophoresis (DE-ACE) method. This method allows the determination of an AT-heparin binding constant (Kd) directly from the cell culture supernatant used to produce the AT variants. Eight measurements per AT variant are sufficient to determine an accurate Kd (uncertainty ≤ 22%, regression coefficient ≥ 0.97), which is not significantly different from the value obtained from a higher number of measurements. Due to the relatively short time required to determine the Kd of one AT variant (2h), this method has the potential for being a low throughput screening method. The method was validated by analyzing five AT variants, whose Kd have been reported in the literature using fluorescence spectroscopy. Finally, the method was applied to estimate the Kd of one new AT variant and one AT conformer, a latent form, that exhibits a significant loss of affinity.
authors: Université Paris Sud, Faculté de Pharmacie, 92290 Châtenay-Malabry, France; CNRS UMR 8612, Institut Galien Paris Sud, 92290 Châtenay-Malabry, France.
authors: Anne-Lise
authors: AL
authors: Marie
authors: Université Paris Sud, Faculté de Pharmacie, 92290 Châtenay-Malabry, France; CNRS UMR 8612, Institut Galien Paris Sud, 92290 Châtenay-Malabry, France.
authors: Nguyet Thuy
authors: NT
authors: Tran
authors: Université Paris Sud, Laboratoire d'Hématologie, EA 4531, 92290 Châtenay-Malabry, France.
authors: Elsa P
authors: EP
authors: Bianchini
authors: Université Paris Sud, Laboratoire d'Hématologie, EA 4531, 92290 Châtenay-Malabry, France.
authors: François
authors: F
authors: Saller
authors: Université Paris Sud, Laboratoire d'Hématologie, EA 4531, 92290 Châtenay-Malabry, France.
authors: Stéphane
authors: S
authors: Pautus
authors: LFB, 91940 Les Ulis, France.
authors: Toufik
authors: T
authors: Abache
authors: LFB, 91940 Les Ulis, France.
authors: Jean-Luc
authors: JL
authors: Plantier
authors: LFB, 91940 Les Ulis, France.
authors: Rémi
authors: R
authors: Urbain
authors: Université Paris Sud, Laboratoire d'Hématologie, EA 4531, 92290 Châtenay-Malabry, France.
authors: Delphine
authors: D
authors: Borgel
authors: Université Paris Sud, Faculté de Pharmacie, 92290 Châtenay-Malabry, France; CNRS UMR 8612, Institut Galien Paris Sud, 92290 Châtenay-Malabry, France. Electronic address: myriam.taverna@u-psud.fr.
authors: Myriam
authors: M
authors: Taverna
mesh_terms: D000991:Antithrombins / Q000737:chemistry*; D018929:Cell Culture Techniques / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D000991:Antithrombins; D006493:Heparin
keywords: Affinity capillary electrophoresis; Binding constant; Fluorescence spectroscopy; Heparin; Recombinant antithrombin