Magnetic Bead-based MAGPIX-Luminex Assay

The Magnetic Bead-based MAGPIX^®-Luminex Assay (MBA) was used to parallel the working steps used in the standard ELISA technique as already described [11 (link),12 (link),17 ]. 2.5 μL aliquots of the mix of microspheres containing 3000 beads per Ag were distributed to individual wells of a white polystyrene opaque round bottom microtiter plate (Ref.103977741, Fisher Scientific, Illkirch, France). 100 μL plasma diluted 1:100 in PBS Tween 0.01% BSA 1% (PBSB) was added in duplicate wells, mixed and incubated with the beads protected from light on a microplate shaker. After removal of plasma and two washing steps with 100μL PBSB, 100μL phycoerythrin-labeled goat anti-human IgG diluted 1:500 (gamma- chain specific, F(ab`)₂ fragment-R-phycoerythrin (Sigma, P-8047 St. Louis, MO) in PBSB was added and incubated in the dark with shaking. The beads were finally resuspended in 120 μL PBSB after two washes and analyzed on a Multiplex MAGPIX system (Millipore, USA) using the xPONENT 4.1 software for acquisition. Antibody responses were expressed in median fluorescence intensity (MFI) per sample as stated by manufacturer’s instructions; individual positivity was considered when the signal was greater than 2 x [mean MFI signal + 3 SD of 6 naïve control sera].