title: Generation of an anti-Dabigatran Monoclonal Antibody and Its Use in a Highly Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Serum Dabigatran.
abstract: BACKGROUND
  Dabigatran (DT) is a direct thrombin inhibitor used to prevent venous and arterial thromboembolism due to atrial fibrillation. DT is the active form of the commercially available prodrug DT etexilate. Although DT has many clinical advantages over warfarin, it increases the incidence of bleeding in patients with renal dysfunction. Circulating levels of DT are increased in such patients because it is mainly eliminated by renal excretion. Therapeutic drug monitoring may therefore help to prevent adverse DT effects, but no method for measuring circulating DT levels has been reported, except for an analysis by liquid chromatography-tandem mass spectrometry. This study sought to develop a novel enzyme-linked immunosorbent assay (ELISA) to measure DT concentrations.
  
  
  METHODS
  Mice were immunized with a DT-keyhole limpet hemocyanin conjugate to generate an anti-DT antibody. Immunized mouse splenocytes and myeloma cells (SP2/0) were fused to obtain an anti-DT monoclonal antibody (DT-mAb). DT-mAb and DT solutions were added to microplate wells coated with a DT-human serum albumin conjugate. DT concentrations were determined based on the principles of ELISA.
  
  
  RESULTS
  DT-mAb was successfully purified from a hybridoma, and the competitive ELISA developed using this DT-mAb could evaluate DT concentrations ranging from 7.8 to 125 ng/mL. The ELISA signal was not linear using DT-spiked serum; however, it was linear when serum ultrafiltrate was used. Weak cross-reactivity with DT etexilate was detected, but no cross-reactivity was observed with other structurally related drugs or drugs commonly used for the treatment of atrial fibrillation.
  
  
  CONCLUSIONS
  The developed competitive ELISA is a valuable and specific tool to analyze free DT in serum ultrafiltrate for therapeutic drug monitoring and pharmacokinetic studies.
authors.affiliation: *Graduate School of Pharmaceutical Sciences; Departments of †Pharmaceutical Health Care Sciences; and ‡Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Oiso
authors.forename: Osamu
authors.initials: O
authors.lastname: Morinaga
authors.forename: Takaaki
authors.initials: T
authors.lastname: Goroku
authors.forename: Takuhiro
authors.initials: T
authors.lastname: Uto
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Shoyama
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kariyazono
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D000069604:Dabigatran / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D000069604:Dabigatran