To overcome the technical difficulties inherent to cell-based neutralization assays, we utilized

an electrochemiluminescence detection method, using the MesoScale Discovery (MSD) platform, to develop non-cell-based assays to assess the presence and nature of anti-IFN-β antibodies. In addition to a screening assay for binding Abs, we developed and characterized a non-cell-based assay, based Selleck AG14699 on the binding of IFN-β to its receptor, to detect and quantify IFN-β NAbs. We present here the first report of the use of a non-cell-based assay for the assessment of NAbs in clinical samples from IFN-β treated patients. Comparative data from this assay and existing cell-based neutralization assays are also shown. Patients sera have been grouped into 3 distinct cohorts (none overlapping), depending on the available

serum samples and the bioassay previously used. Cohort A (n = 46) includes post-treatment only samples, tested in the MxA protein assay. Cohort B (n = 10) includes post-treatment only samples, tested in the antiviral assay. Cohort C (n = 31) includes sequential samples (baseline and subsequent time-points), tested in antiviral and reporter gene assays. Pooled or individual sera from normal healthy CFTR activator donors were also included in the study (n = 27). Ethical approval and informed consent from patients and donors were obtained in accordance with the guidelines in the Helsinki Declaration for all sera tested. Therapeutic grade IFN-β-1a preparations were supplied to NIBSC directly by the manufacturer and routine supply chains. The recombinant human IFN-α/β R2/Fc chimera was obtained Avelestat (AZD9668) from R&D Systems (4015-AB) and the recombinant vaccinia virus-encoded neutralizing type I interferon receptor B18R, was obtained from eBioscience (14-8185). A lyophilized pooled human serum sample from IFN-β treated patients (coded 99/606, available from NIBSC) and a hyperimmune sheep polyclonal anti-IFN-β serum generated at NIBSC served as positive controls. IFN-β-1a was biotinylated with EZ-link

Sulfo-NHS-LC-Biotin (Thermo Scientific, 21335). The biotin, resuspended in water, was mixed with IFN-β to give a molecular challenge ratio of label/protein of 2 and the mixture left at room temperature for 1 h to enable the labeling reaction to reach completion. The labeled IFN-β was isolated using a PD-10 desalting column (GE Healthcare, 52-1308-00 AP), recovered in PBS and stored at 4 °C. The labeling of IFN-β-1a with ruthenium-NHS-ester was performed as per manufacturer’s instructions. Briefly, the ruthenium-NHS-ester (labeled Sulfo-Tag, MSD, R91AN-1) was resuspended in cold distilled water and mixed with IFN-β to give a molecular challenge ratio of label/protein of 2. The reaction mixture was incubated for 2 h at room temperature and the conjugated IFN-β isolated by centrifugation using Amicon Ultra 3K centrifugal filters (Millipore, UFC800324), recovered in PBS and stored at 4 °C.