Cell-based reporter gene assay for therapy-induced neutralizing antibodies to interferon-beta in multiple sclerosis.

Patients with therapy-induced neutralizing antibodies (NAbs) to interferon-beta (IFN-ß) have reduced responses to IFN-ß treatment, resulting in higher relapse rates, increased magnetic resonance imaging activity, and a higher risk of disease progression. A functional assay was employed for both screening and titering of IFN-ß NAbs utilizing a human cell line transfected with a luciferase reporter gene responsive to IFN-ß. This assay demonstrated 100% sensitivity and specificity compared with the traditional cytopathic effect (CPE) assay and normal donor specimens. Additionally, 183 patients with multiple sclerosis (MS) undergoing therapy with IFN-ß were tested in the reporter gene assay. Percent positivity for NAbs to the IFN-ß was as follows: Avonex (1α) 26.5%, Rebif (1α) 34.1%, and Betaseron (1ß) 31.8%. The IFN-ß reporter gene assay showed excellent correlation with the well-established CPE assay offering clear advantages. The 50% false-positivity rate typically seen in enzyme-linked immunosorbent assays could be eliminated by using a functional assay for both screening and titering. Results can be reported within 20 h, and the cell line is cryopreserved, eliminating the need to maintain live viral and cell cultures. The use of this functional assay should be a valuable tool for detecting and monitoring the presence of NAbs in IFN-ß-treated patients with MS.

Analysis of proinflammatory and anti-inflammatory cytokine serum concentrations in patients with multiple sclerosis by using a multiplexed immunoassay.

We examined cytokines and other inflammatory markers in serum samples from 833 patients with multiple sclerosis and 117 healthy control subjects. A multiplexed immunoassay was used to assess the concentrations of 13 cytokines/inflammatory markers: interferon (IFN)-γ; interleukins (ILs)-1ß, 2, 4, 5, 6, 8, 10, 12, and 13; tumor necrosis factor (TNF)-α; IL-2 receptor; and soluble CD40 ligand. Significant increases between patients and control subjects were found for IFN-γ (mean, 7.5 vs 0.4 pg/mL; P = .0002), IL-2 (mean 5.7 vs 1.0 pg/mL; P =.0002), IL-1ß (mean, 23.0 vs 11.3 pg/mL; P ≤ .0001), TNF-α (mean, 4.1 vs 1.2 pg/mL; P = .01), IL-4 (mean, 1.4 vs 0.1 pg/mL; P ≤ .0001), IL-10 (mean, 16.8 vs 7.5 pg/mL; P = .03), and IL-13 (mean, 4.5 vs 0.8 pg/mL; P ≤ .0001). Profiling cytokines in multiple sclerosis may help to identify mechanisms involved in the pathogenesis of the disease, aid in monitoring the disease course and in evaluating responses to specific therapies, and, potentially, lead to new therapies directed at cytokines or their receptors.

Serum anti-GAGA4 IgM antibodies differentiate relapsing remitting and secondary progressive multiple sclerosis from primary progressive multiple sclerosis and other neurological diseases.

The serum level of IgM antibodies against Glc(alpha1,4)Glc(alpha) (GAGA4) is higher in relapsing remitting multiple sclerosis (RRMS) compared to other neurological disease (OND) patients and healthy controls (HC). Detecting the level of anti-GAGA4 antibody by enzyme immunoassay and total IgM, we confirmed that anti-GAGA4 IgM can differentiate RRMS from OND patients and HC. Moreover, secondary progressive MS (SPMS) and RRMS patients have similar levels of anti-GAGA4 demonstrating the biomarker's presence throughout the disease. Interestingly, the anti-GAGA4 assay may also differentiate between primary progressive MS (PPMS) and RRMS/SPMS patients, since nearly all PPMS patients were negative for the assay.