Neutralizing antibodies to interferon beta in multiple sclerosis: Analytical evaluation for validation of a cytopathic effect assay Catherine Massart a, ⁎ , Jacqueline Gibassier a , Joël Oger b , Emmanuelle Le Page c , Gilles Edan c a Laboratoire d'Hormonologie-Marqueurs, CHU Ponchaillou, 35033 Rennes, France b Department of Neurology, Pathology and Neurosciences, University of British Columbia, Vancouver BC, Canada V6T 2B5 c Service de Neurologie, CHU Ponchaillou, 35033 Rennes, France Received 25 August 2006; received in revised form 22 September 2006; accepted 23 September 2006 Available online 30 September 2006 Abstract Background: Recent guidelines have recommended the use of validated assays for the measurement of neutralizing antibodies (NABs) to interferon beta (IFNβ) in patients with multiple sclerosis (MS). In an attempt of validation, we studied the analytical performance of a bioassay based on antiviral cytopathic effect (CPE) using WISH cells and the vesicular stomatitis virus (WISH/VSV CPE). Methods: NAB titres measured with the WISH/VSV CPE assay in 63 sera from IFNβ-treated MS patients were compared to those obtained with the reference CPE method using A549 cells and the encephalomyocarditis virus. Binding antibodies (BABs) were measured using a capture ELISA as a screening test for NABs. Results: No false-negative BAB was obtained in our patients. The between-run coefficients of variation (CVs) determined with log 10 titres of the NIH anti-IFNβ (G038-501-572) yielded good results (≤ 10.4%) and within-run variability was excellent (CV ≤ 2%). The log 10 titres obtained with both CPE assays were highly correlated (r = 0.969 and r = 0.884 for anti-IFNβ-1a and anti-IFNβ-1b, respectively). The same patients were found NAB-positive with both CPE assays. Conclusion: Because of its good precision, sensitivity and excellent correlation with the reference CPE method, the WISH/VSV CPE bioassay can be used in the follow-up of IFNβ-treated MS patients. © 2006 Elsevier B.V. All rights reserved. Keywords: Interferon beta; Neutralizing antibodies; Multiple sclerosis treatment; Cytopathic effect method; NIH References Reagent G038-501-572 1. Introduction Treatment with interferon beta (IFNβ) is a first-line therapy for relapsing-remitting multiple sclerosis (MS), significantly reducing the frequency of clinical attacks, lesion load and disability [1–4]. Recombinant IFNβ-1a or IFNβ-1b is currently used for the treatment of MS. However, some patients develop neutralizing antibodies against IFNβ (NABs) within six to eight months after initializing treatment [2,5,6] resulting in a loss of clinical efficacy of IFNβ [3,6–8]. IFNβ antibodies are detected in patient sera either by their capacity for binding IFNβ (BAB: binding antibodies) or more importantly by their neutralizing effects (NAB) [9]. Among BAB assays, Western blot methods and direct ELISA methods were rejected by the European Federation of Neurological Societies (EFNS) recommendations due to high rate of false-negative or false-positive results [10]. In contrast, radioimmunoprecipitation assay [11] and capture ELISA methods [12–14] are fast and easily performed by laboratories and were recommended for IFNβ antibody screening before performing NAB measurement [10]. Almost all reported NAB assays in the literature used either IFNβ- induced myxovirus A (MxA) gene production [5,15–17] or IFNβ-induced cell protection from the viral cytopathic effect Clinica Chimica Acta 377 (2007) 185 – 191 www.elsevier.com/locate/clinchim Abbreviations: BAB, binding antibody; CI, confidence interval; IFNβ, interferon beta; CPE, cytopathic effect; CV, coefficient of variation; EFNS, European Federation of Neurological Societies; ELISA, enzyme-linked immunosorbent assay; EMC, encephalomyocarditis; IgG, immunoglobulin G; IU, international units; LU, laboratory unit; NAB, neutralizing antibody; MAb, monoclonal antibody; MS, multiple sclerosis; MxA, myxovirus A; MTT, 3-(4,5- dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; OD, Optical Density; PBS, phosphate buffer saline; SD, standard deviation; TRU, ten-fold reduction unit; VSV, vesicular stomatitis virus; WISH, Wistar Institute Susan Hayflick. ⁎ Corresponding author. Tel.: +33 2 992 84271; fax: +33 2 992 84145. E-mail address: catherine.massart@chu-rennes.fr (C. Massart). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.09.021