Anti human igg biotin

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.

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.

Measurement of plasma IgG antibodies was performed by multiplex suspension array using the Luminex technology, as described [46 (link)]. MagPlex magnetic carboxylated microspheres (Luminex Corporation, TX, USA) were covalently coated with 3 μg of protein/peptide per 1.1–1.4 million beads following manufacturer’s instructions. 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. Circa 1000 beads per analyte were incubated with plasma (1:100 dilution) in duplicates, and subsequently with anti-human IgG-biotin (Sigma-Aldrich), followed by streptavidin-conjugated R-PE (Fluka, Madrid, Spain). Beads were acquired on the BioPlex100 system (Bio-Rad, Hercules, CA), and results expressed as median fluorescence intensity of duplicates. Value against GST alone was subtracted for VIR proteins. Raw GST values are presented in S1 Fig. Cross-reactivity was ruled out in a pilot study analyzing a subset of plasmas in singleplex and multiplex. Samples in IN were analyzed with identical protocols and instruments.