title: Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta.
abstract: Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low positive titers needs to be further evaluated.
authors.affiliation: Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Christina
authors.initials: C
authors.lastname: Hermanrud
authors.affiliation: Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Ryner
authors.affiliation: Department of Neurology, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Luft
authors.affiliation: Neuroimmunology Laboratory, DMSC, Department of Neurology, Rigshospitalet, Region H, Copenhagen, Denmark.
authors.forename: Poul Erik
authors.initials: PE
authors.lastname: Jensen
authors.affiliation: Department of Neurology, Medical Faculty, University of Düsseldorf, Germany.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Ingenhoven
authors.affiliation: Sanofi-Aventis, Deutschland GmbH, Germany.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Rat
authors.affiliation: Department of Neurology, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Florian
authors.initials: F
authors.lastname: Deisenhammer
authors.affiliation: Neuroimmunology Laboratory, DMSC, Department of Neurology, Rigshospitalet, Region H, Copenhagen, Denmark.
authors.forename: Per Soelberg
authors.initials: PS
authors.lastname: Sørensen
authors.affiliation: INSERM 996, University Paris-Sud, Paris, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Pallardy
authors.affiliation: GlaxoSmithKline, BioPharm Research and Development, King of Prussia, PA, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Sikkema
authors.affiliation: Merck Serono NBE Bioanalytics, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Bertotti
authors.affiliation: Sanofi-Aventis, Deutschland GmbH, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kramer
authors.affiliation: Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary, University of London, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Creeke
authors.affiliation: Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden. Electronic address: Anna.Fogdell-Hahn@ki.se.
authors.forename: Anna
authors.initials: A
authors.lastname: Fogdell-Hahn
mesh_terms: D057134:Antibodies, Neutralizing / Q000097:blood*; D001681:Biological Assay; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D017930:Genes, Reporter; D006801:Humans; D016899:Interferon-beta / Q000276:immunology*; D008156:Luciferases; D009103:Multiple Sclerosis / Q000188:drug therapy / Q000276:immunology; D009500:Neutralization Tests; D012680:Sensitivity and Specificity
chemical_list: D057134:Antibodies, Neutralizing; D016899:Interferon-beta; D008156:Luciferases
keywords: Anti-drug antibodies; Bioassay; Interferon beta; Luciferase; Multiple sclerosis; Neutralizing antibodies