Research paperValidating parameters of a luciferase reporter gene assay to measure neutralizing antibodies to IFNβ in multiple sclerosis patients
Introduction
Interferon beta (IFNβ) is a first line treatment for Multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system (CNS) characterized by inflammatory demyelination. Therapy with IFNβ reduces disease exacerbations and magnetic resonance imaging (MRI) burden (The IFNβ Multiple Sclerosis Study Group and the University of British Columbia MS/MRI Analysis Group, 1996, Giovannoni et al., 2002). However, in a proportion of patients on therapy, binding antibodies (BAbs) and neutralizing antibodies (NAbs) occur. NAbs reduce bioavailability of IFNβ to its receptors and high NAb titers reduce the efficacy of the drug. NAb positive patients may have higher relapse rates (Boz et al., 2007), more disease progression and more new or enlarging MRI lesions in comparison to NAb negative patients (Francis et al., 2005).
BAbs and NAbs are defined by the assay method used. BAbs are measured by binding assays such as enzyme linked immunosorbent assays (ELISA), radioimmunoprecipitation assays (RIPA), or column chromatography assays, which determine the presence of antibodies that bind to IFNβ. NAbs are defined by inhibition of in vitro bioassays that measure the activity of IFNβ in the presence of sera containing NAbs. Two common assays are available to measure NAbs. The cytopathic effect (CPE) assay measures the antiviral activity of IFNβ upon challenge of a cell line with a virus. The myxovirus resistance protein A (MxA) induction assay measures IFNβ inducible gene product MxA protein or MxA mRNA to quantify IFNβ response (Files et al., 1998, Pungor et al., 1998, Bertolotto et al., 2007). Because these methods of NAb detection are ardous, a BAb assay can be used to screen for further NAb testing (Sorensen et al., 2005). There is a need for a new, specific, sensitive and less arduous assays for quantifying NAbs in IFNβ treated MS patients. Recently, reporter gene assays have been described to measure NAbs (Farrell et al., 2008, Lallemand et al., in press).
We report the validation of a luciferase reporter gene assay to detect NAbs based on the activation of the early IFN inducible 6–16 promoter by IFN, described by R. Farrell and G. Giovannoni (Farrell et al., 2008). This method uses the luminescence signal from cell line HL 116 which is the human fibrosarcoma cell line HT1080 transfected with a plasmid containing a luciferase cDNA controlled by the immediate early IFN inducible 6–16 promoter (Uze et al., 1994). 6–16 is a gene specific only to type I interferons (Der et al., 1998). When IFNβ binds to receptor, luciferase is produced and the response to IFNβ can be quantified by a luminometer upon the addition of substrate. Patients' sera are serially diluted and incubated with a constant amount of IFNβ (10U/mL). In the presence of NAbs, less luciferase is produced. Results are reported in Tenfold Reduction Units (TRU)/mL (Grossberg et al., 2001) as per WHO recommendations. We validated this assay by comparing NAb status and NAb titers obtained on split samples with the reference A549/EMCV CPE considered gold standard (reference: Sorensen et al., 2005) assayed at the Medical College of Wisconsin (Grossberg et al., 1986).
Section snippets
Sera
Sera were selected from the University of British Columbia MS serum bank on the following criteria; a) treated with a single IFNβ product, b) availability of antibody results from the routine BAb testing by the sandwich ELISA and when positive were further tested with the A549/EMCV CPE method by S. Grossberg (Grossberg et al., 1986). Sera were collected more than 12 hours after the last interferon injection, a time when circulating levels of injected interferon are below threshold detectable by
Plate color
Three types of sterile, tissue culture plates were tested for use in the luciferase assay: 96 well white plates with a transparent bottom (6005181, PerkinElmer, USA), black plates with a transparent bottom (Costar 3603, NY, USA), and black plates with a white bottom (6005688, PerkinElmer, USA). White plates yielded higher counts, but light contamination between neighboring wells was observed. Higher counts were observed for the black plates with the white bottom, on a log–log transformation,
Discussion
In this study the R. Farrell and Giovannoni luciferase assay methodology was validates for the detection of NAbs to IFNβ. We found that the parameters that optimize assay sensitivity and reproducibility include using a 96-well black plate with a transparent bottom allowing the visual inspection of cells, seeding 40,000 cells/well allowing high sensitivity to IFNβ with low coefficients of variation between wells, incubating plates 5 h permitting a one-day rapid assay without compromising assay
Acknowledgements
We thank Dr. G. Uzé (Montpellier, France) for the generous gift of the cell line transfected with the reporter gene HL116. This study was supported by grants-in-aid from Bayer, Biogen Idec, EMD Serono and Teva Neurosciences and by the Christopher Foundation (Vancouver).
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These 2 co-authors contributed equally