title: Analysis of neutralizing antibodies to therapeutic interferon-beta in multiple sclerosis patients: a comparison of three methods in a large Australasian cohort.
abstract: Persistent high-titre neutralizing antibodies (NAB) to therapeutic interferon-beta(IFNbeta)in multiple sclerosis patients reduce therapeutic efficacy. Difficulties in standardization of cell-based bioactivity assays have hindered interlaboratory comparison of NAB titres and the determination of a clinically relevant definition of seropositivity. We determined NAB status in Australasian multiple sclerosis patients receiving IFNbetausing both the antiviral cytopathic effect (CPE) assay (n = 227) and the more specific ELISA for the type I interferon-inducible MxA protein (n = 350). While the log(10) titres determined in the two assays were highly correlated (p < 0.0001; r = 0.967) with similar distributions, the MxA assay was more sensitive, detecting lower concentrations of NAB than the CPE assay. The range of titres determined in the CPE assay was 10 to >7290; and 9 to 53,700 in the MxA assay, with ranked titre distribution highlighting the arbitrary nature of currently accepted definitions of NAB seropositivity. Bioactivity of injected IFNbetawas significantly reduced in NAB-positive patients (p = 0.006; NAB MxA titres = 184 to 5340) compared to NAB-negative patients as assessed ex vivo using real-time RT-PCR analysis of MxA gene induction. The range of MxA mRNA levels in healthy controls was remarkably consistent with previously published results, regardless of the assay standardization method [Gilli, F., Sala, A., Marnetto, F., Lindberg, R.L., Leppert, D. and Bertolotto, A. (2003) Comparison of IFNbeta bioavailability evaluations by MxA mRNA using two independent quantification methods. Abstract, ECTRIMS Meeting, Milan, Italy; Pachner, A., Narayan, K., Price, N., Hurd, M. and Dail, D. (2003a) MxA Gene Expression Analysis as an Interferon-beta Bioactivity Measurement in Patients with Multiple Sclerosis and the Identification of Antibody-Mediated Decreased Bioactivity. Mol. Diagn. 7, 17-25]. Assessment of IFNbetaresponse ex vivo accounts for both circulating factors and the cellular response to IFNbeta, and the data support the development of the MxA gene induction assay for the routine screening of patients receiving IFNbeta.
authors.affiliation: Institute for Immunology and Allergy Research, Westmead Millennium Institute, University of Sydney, Westmead Hospital, Australia.
authors.forename: Fiona
authors.initials: F
authors.lastname: McKay
authors.forename: Stephen
authors.initials: S
authors.lastname: Schibeci
authors.forename: Robert
authors.initials: R
authors.lastname: Heard
authors.forename: Graeme
authors.initials: G
authors.lastname: Stewart
authors.forename: David
authors.initials: D
authors.lastname: Booth
mesh_terms: D000906:Antibodies / Q000276:immunology*; D044362:Australasia; D045744:Cell Line, Tumor; D015331:Cohort Studies; D003588:Cytopathogenic Effect, Viral / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016899:Interferon-beta / Q000276:immunology / Q000627:therapeutic use*; D020529:Multiple Sclerosis, Relapsing-Remitting / Q000188:drug therapy* / Q000276:immunology*; D064547:Myxovirus Resistance Proteins; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018709:Statistics, Nonparametric
chemical_list: D000906:Antibodies; C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D012333:RNA, Messenger; D016899:Interferon-beta; D019204:GTP-Binding Proteins