Autoantibody Profiling in Serum Samples

Serum ANA levels were measured by ELISA (Inova, San Diego, CA, USA) using the manufacturer's suggested cut-off of >20 units to define positive results. Other ELISA kits were used to measure CCP antibodies (Inova), thyroglobulin autoantibodies (Genway Biotech Inc., San Diego, CA, USA) and total IgG (Bethyl Laboratories Inc., Montgomery, TX, USA). The extractable nuclear antibody (ENA) panel for eight additional specificities was a Luminex-based multiplex assay, and positive results were as defined by the manufacturer (Inova). Serum reactivity to a panel of approximately 101 autoantigens and 6 controls was measured on a slide-based array that has been described previously (Additional file 1) [6 (link)]. Serum samples (1 μL, diluted 1:100) were added to each array in duplicate and autoantibodies were detected with Cy3-labelled anti-human IgG and Cy-5 labeled anti-human IgM simultaneously. Images were generated for analysis and mean fluorescence intensities (MFI) were determined as previously described. Heat maps were generated using Cluster and Treeview software (Michael Eisen, Berkeley CA, USA) [7 ]. On the heat map, intensities higher than the row mean are colored red, those below the mean are green and cells with signals close to the mean are black. Gray was used to denote missing data.