Multiplex magpix system

The custom magnetic bead-based MAGPIX^®-Luminex Assay (MBA), adapted to parallel the steps used in the standard ELISA technique has been previously described [24 (link), 26 (link), 27 ]. The mix of microspheres was kept in an opaque vial. 2.5 µL aliquots containing 1000 beads per Ag were dispensed to individual wells of a white polystyrene opaque round bottom microtiter plate (Ref.103977741, Fisher Scientific, Illkirch, France). 100 μL of plasma diluted 1/200 in PBS, 0.01% Tween, 1% BSA (PBSB) was added to duplicate wells, mixed and incubated protected from light at room temperature on a microplate shaker (IKA^®MTS, Wilmington, NC) at 350 rpm for 45 min. After washing twice, 100 μL of phycoerythrin-labelled 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 45 min in the dark with shaking at 350 rpm. The beads were then re-suspended in 120 μL PBSB and analyzed on a Multiplex MAGPIX system (Millipore, USA) using the xPONENT 4.1 software for data acquisition. Antibody responses were expressed in median fluorescence intensity (MFI) per sample as stated by manufacturer’s instructions; readings were considered positive when the signal was greater than the mean MFI signal + 3 SD of 6 naïve control sera (a pool of non-immune sera from blood donors living in France).

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].

Measurements were performed using the Multiplex MAGPIX system (Millipore Corporation, Billerica, MA, USA) using the XPONENT 4.2 software for acquisition and assay Interleukin-1 [IL-1], interleukin-10 [IL-10], interleukin-17 [IL-17] and Tumor Necrosis Factor-Alpha [TNF-α] plasma/serum levels were assayed using Kit Cytokine Milliplex Map Rat Cytokine/Chemokine Magnetic Bead Panel design.