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The current study presents a western blot assay showing good sensitivity (81%) and specificity (97%) for detecting serum antibodies to Aquaporin-4 (AQP4) in patients with Neuromyelitis Optica (NMO). By using western blot, we were able, for the first time, to distinguish serum immunoreactivity against either of the two AQP4 isoforms, showing that antibodies recognizing the linear AQP4-M1 isoform are specific for NMO. Moreover, the high sensitivity and specificity of the western blot assay in detecting serum anti-AQP4 antibodies in NMO patients suggest that such auto-antibodies may be of linear nature, thus recognizing epitopes that are displayed in denatured protein.

Therapeutic proteins like recombinant human interferon-beta (rhIFNbeta) may induce neutralising antibodies (NABs), which inhibit their efficacy. Hence, there is a great need for strategies to predict whether a formulation will induce an immune response. Immune tolerant transgenic animals are important tools to study this phenomenon. This article describes a bioassay for NABs detection in mouse sera. The bioassay corresponds to the MxA Gene expression Assay (MGA) for human sera and measures the inhibition by mouse serum of the IFNbeta induced MxA mRNA. Samples from 6 non-immunised and 14 IFNbeta-immunised mice were tested for both binding antibodies (BABs) and NABs using the bioassay. All 16 mouse sera tested positive for NABs were also positive for BABs; BAB and NAB levels were correlated with a coefficient of 0.62 (p=0.0186). The intra-assay variations ranged from 1.38% to 5.26% (mean 3.03%). Effects of cytotoxicity against A549 cells were slightly evident at low serum dilutions (i.e. 1/10, 1/20), but levels of damaged cells were easily evaluated based on the threshold cycle (Ct) values of the housekeeping gene 18SrRNA. The possibility of measuring NABs, in addition to BABs, in mouse sera increases the usefulness of the animal model, in studying the many factors influencing the immunogenicity of rhIFNbeta.

Many multiple sclerosis (MS) patients treated with interferon-beta (IFNbeta) develop anti-IFNbeta antibodies (BAbs), which can interfere with both in vitro and in vivo bioactivity of the injected cytokine. Objective of this study was to correlate these measures. Among the 256 enrolled patients, 11 (4.3%) showed a significant inhibition of the IFNbeta activity in vitro, but no measurable BAbs. As a whole, in vivo bioactivity was inhibited in 9/11 (82%) of these patients. A minority of IFNbeta treated patients have a non-antibody mediated neutralising activity, which competitively inhibits the bioactivity both in vitro and in vivo.

There are two commonly employed types of bioassays for the detection of neutralizing antibodies (NAbs) against interferon-beta (IFNbeta): the cytopatic effect assay (CPE), and the MxA (myxovirus resistance protein A) protein assay (MPA). This article describes a bioassay based on the real time PCR measurement of mRNA that results from the induction, in cultured human cells, of the MxA gene by IFNbeta. Serum samples from 104 patients with multiple sclerosis (MS) treated with IFNbeta were tested for NAbs using our real time PCR bioassay. NAbs also were measured in the same specimens by the MPA assay and CPE assay. The calibration range of the real time PCR bioassay is 0.125-30 LU/mL. The range of the intra- and inter-assay variations (coefficients of variation in log(10)) were 4.05% (range 0.88%-7.90%) and 4.42% (range 0.31%-9.15%), respectively. Samples of the three commercial preparations of IFNbeta-1a and -1b were measured showing dose-response curves parallel to that of the NIH reference IFNbeta (mean SD at the midpoint of the dose-response curve=5%). In addition, the assay was robust with respect to number of cells plated (i.e., increasing cell densities from 12x10(3)/well to 384x10(3)/well resulted in 3.03% variability in MxA expression normalized with glyceraldehyde-3 phosphate dehydrogenase). NAbs titers measured were closely comparable to those obtained by the MPA [r(spearman)=0.899; 89% of observed agreements; K=0.779] and the CPE [r(spearman)=0.7899); 86%; K=0.729] assays. Despite the obvious disadvantage of cost, when carried out according to quality assurance guidelines for molecular diagnostics the new MxA gene-expression assay (MGA) has significant advantages over the other methods for testing NAbs: it has excellent reliability and reproducibility, and utilizes equipment and methodologies already accessible in many clinical laboratories.

Relapsing-remitting multiple sclerosis (MS) has a very fluctuating course and responsiveness to interferon beta (IFN-beta) treatment in each patient is extremely difficult. Agreement exists about the negative role of neutralising antibodies (NAbs) on clinical efficacy and markers of IFN-beta bioavailability have been studied; no guidelines exist yet about what to do when a patient becomes NAbs positive or IFN biological activity is lost. In this study 405 MS patients have been longitudinally studied for NAbs and MxA expression. A spontaneous disappearance of NAbs was observed in a few patients with low antibody titre; according to the clinical course, a therapeutic modification has been made in patients persistently NAbs positive; in these patients NAbs persisted over time despite the interruption of IFN therapy.

Background: MxA is an antiviral protein exclusively induced by type I interferons (IFN) and some viruses, and MxA gene expression is one of the most appropriate markers for measuring the biologic activity of exogenous IFNbeta., Methods: A new quantitative-competitive PCR method was used to quantify MxA mRNA in peripheral blood mononuclear cells of 99 treatment-naïve and 92 IFNbeta-treated patients with MS (22 Avonex, 17 Betaferon, and 53 Rebif-22). Every 3 months, IFNbeta-induced neutralizing antibodies (NAb) were evaluated in sera using a cytopathic effect assay. Three categories of patients were identified: NAb negative (NAb-), persistent NAb positive (NAb+, >or=2 consecutive positive samples), and isolated NAb+ (one positive sample)., Results: Treatment-naïve patients expressed detectable MxA mRNA levels (mean = 36 +/- 32 fg MxA/pg glyceraldehyde-3-phosphate dehydrogenase (GAPDH); range 1 to 160) and an upper normal threshold was established (mean + 3 SD = 132 fg MxA/pg GAPDH). IFNbeta-treated patients exhibited more than 11-fold higher levels (mean = 412 +/- 282 fg MxA/pg GAPDH; range 16 to 1,172). However, 17 patients did not exhibit an increase in MxA mRNA level; 15 of these 17 patients showed a concurrent Nab+ titer. Moreover, 13 were persistent NAb+. Isolated NAb+ patients did not show a decrease in bioavailability of IFNbeta (n = 9; mean = 567 +/- 366 fg MxA/pg GAPDH; range 83 to 1,120). In NAb- patients, bioavailability was comparable among the three different IFNbeta preparations 12 hours after injection., Conclusion: During IFNbeta therapy, the presence of NAb reduced or abolished bioavailability in a relevant percentage of patients. These data could be important for the early detection of patients with MS who are not responsive to IFNbeta therapy.