Evaluation of 4 different assays to determine antibodies to interferon-beta

Aim of the study: Betainterferons (IFN-beta) are the most widely used drugs to treat multiple sclerosis (MS). However a significant proportion of patients develop neutralizing antibodies (NABs) to IFN-beta, which may interfere with treatment efficacy. Many different assays are in use to detect and quantify antibodies to IFN-beta and their neutralizing activity. However, systematic comparative analyses, which address the inter assay-variation, are largely missing.

Methods: Here we report the results of a blinded comparative analysis of 4 assays evaluated with 250 blinded samples in two different centers. A westernblot and capture (c) ELISA were used to determine binding antibodies to IFN-beta. The biological activity of the antibodies was investigated by the cell based in vitro MxA induction assay. In addition MxA mRNA levels in blood 12 hrs after IFN-beta injection were studied by rt-PCR (in vivo MxA assay). The laboratory in Innsbruck performed the westernblot and in vivo MXA assay, the center in Düsseldorf the cELISA and in vivo MxA assay.

Results: The study demonstrated that the westernblot screening assay had a high sensitivity but lacks specificity for neutralizing antibodies. By contrast, the cELISA had a slightly lower sensitivity but detected neutralizing antibodies with high specificity. In patients with neutralizing antibodies we found a strong correlation between titers obtained by the cELISA, MxA in vitro and the in vivo assays. NAB testing with the combination of westernblot/in vitro MxA assay and cELISA/in vivo MxA assay revealed consistent results in 96% of the samples. Discordant results were obtained with sera, which contained low titers of NABs.

Conclusions: This was the first large scale comparative study evaluating different new and established methods to measure antibodies to IFN-beta. Although the assays used in the study differed substantially, they obtained almost identical results with respect to NAB titers.