Multiplex Plasma IgG Antibody Measurement

Measurement of plasma IgG antibodies was performed by multiplex suspension array using the Luminex™ technology, as described before (24 (link)). Briefly, 1.1–1.4 million MagPlex^® magnetic-carboxylated microspheres (Luminex Corporation, TX, USA) with different spectral signatures were covalently coated with 3 µg of each protein/peptide, following the manufacturer’s instructions. Protein-coupled beads were quantified in a Guava^® Flow Cytometer (Millipore) and mixed in equal amounts. A unique batch of microspheres was prepared for the whole study, including the samples analyzed in IN. Approximately 1,000 beads per analyte were incubated with each plasma (1:100 dilution) in duplicates, and subsequently with antihuman IgG-biotin (Sigma-Aldrich), followed by streptavidin-conjugated R-PE (Fluka, Madrid, Spain). Beads were acquired on the BioPlex100 system (Bio-Rad, Hercules, CA, USA), and results were expressed as median fluorescence intensity (MFI) of duplicates. Value against GST alone was subtracted from correspondent proteins. Raw GST data have been previously published (24 (link)). Cross-reactivity was ruled out in a pilot study analyzing a subset of plasmas in singleplex and multiplex (not shown). Samples in IN were analyzed with identical protocols and instruments.