, the presence of polyclonal or monoclonal Ig specific for glucosylsphingosine (GlcSph, or LGL1) was performed using an immunoblotting assay adapted from Nair et al, vol.8

, Polyvinylidene fluoride (PVDF) membranes were incubated for 90 min in 100 ?g/mL of GlcSph in 0.1 M sodium bicarbonate, rinsed 3 times in PBS and 0.1% Tween 20 detergent, and then blocked for 2 h with 5% bovine serum albumin (BSA) in PBS and 0.1% Tween 20. Samples of serum or purified monoclonal IgG or IgAs were submitted to agarose gel electrophoresis, and then the gels were blotted onto the GlcSph-saturated membranes by diffusion blotting during 12 min, GlcSph (ref. 2086, with purity assessed at >98% by thin-layer chromatography) was purchased from Matreya LLC/Cayman Chemical

, The MIAA Assay As previously published, the MIAA assay allows testing for panels of commercially available antigens or/and lysates from EBV, herpes simplex virus 1 (HSV-1), HSV-2, cytomegalovirus (CMV), varicella zoster virus (VZV), HCV, Helicobacter pylori (H. pylori), Toxoplasma gondii (T. gondii), and Borrelia burgdorferi (B. burgdorferi

M. D. Columbia, U. Immunodiag-;-columbia, M. D. , U. ). , and E. Bio, The arrays consist of 8 × 8 matrices that included: (i) 13 Ag: 2 for EBV, 3 for HCV, 1 for T. gondii, 1 for H. pylori, 2 for HSV-1, 2 for HSV-2, 2 for VZV; (ii) 5 lysates: CMV, T. gondii, H. pylori, HSV-1, and HSV-2; (iii) 2 mixes: one of 5 CMV Ag, and one of 2 B. burgdorferi Ag. For hybridization, Ig concentrations were adjusted to 400 ?g/mL for serum and from 50 to 200 ?g/mL for purified monoclonal Igs. 80 ?L of samples were incubated for 2 h at room temperature. After washing, slides were incubated with a labelled secondary antibody (0.4 ?g/mL Dylight TM 680 Labelled Goat anti-human IgG (H+L), from Sera Care, or DyLight TM 680 goat anti-human IgA? chain from ImmunoReagent

, Microarray Acquisition & Analysis Software (Molecular Devices

, Analysis of IgG sialylation Whenever possible, for each patient, samples from preparations of non-clonal Igs and of purified monoclonal Ig were studied in parallel

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