Multiplex Antibody Profiling using Luminex

Measurement of plasma IgG antibodies was performed by multiplex bead-based assay using the Luminex technology, as described (Rui et al., 2011 (link)). The different recombinant proteins and peptides included in this study were covalently coupled to four MagPlex magnetic carboxylated microspheres (Luminex Corporation, TX, USA). Next, in 1.25 million coated beads we coupled 1 μg of each antigen following manufacturer’s instructions. Using a Neubauer chamber coated-beads were quantified and mixed in equal amounts. Only one batch of microspheres was prepared for the study. Plasma (1:100 dilution) was incubated with around 2000 beads per analyte in duplicates, followed by anti-human IgG-biotin (1:4000 dilution) (Sigma-Aldrich) incubation. Next, streptavidin-conjugated to R-phycoerthrin (R-PE) (1 μg/mL) was incubated for 10 min at RT and beads were acquired on the BioPlex100 system (Bio-Rad, Hercules, CA) and express the results as median fluorescence intensity (MFI) of duplicates. Cross-reactivity was ruled out analyzing a subset of plasmas in singleplex and multiplex. A panel of eight negative controls from healthy individuals from Barcelona was included on every plate. Cutoffs for antibody positivity were determined for each plate by calculating mean +2 standard deviation of the negative control values.