Autoimmune, Infectious Disease Multiplexing Tool Figure 3. Cross-reactivity of ganglioside antibodies. GM1- and GA1-conjugated beads showed considerable cross reactivity with anti-GD1b sera since these gangliosides have common terminal sugar sequence (panels b, c; dotted red square in panel e). GM1 beads displayed cross- reactivity with anti-GM2 serum due to the shared epitope groups in the inner part of the GM1- and GM2-gangliosides (panel d; dotted black square in panel e). Cross-reactivity was estimated comparing the reporter signal from the beads coated with the ganglioside related to the tested serum and the signals from the beads coated with other gangliosides. doi:10.1371/journal.pone.0042681.g003 instrument can be expected in diagnosis and monitoring of the neuropathic or neurological disorders, and in investigating their possible relations with infectious diseases or vaccination. Materials and Methods Fabrication of beads coated with gangliosides All gangliosides were purchased from American Research Products, INC. For bead array fabrication, fluorescent carboxyl- ated non-magnetic beads (Luminex) were used, #45 for GM1 ganglioside, #27 for GM2 ganglioside, #25 for GA1 asialo ganglioside, #14 for GD1b diasialo ganglioside. First, 50 ml of bead suspension (from stock 1.25*107 beads/ml) was diluted in 270 ml of 100 mM, pH 3.5 MES (2-(N-morpholino)ethanesulfonic acid) buffer containing 0.05% Tween-20 (MES-T, all components from Sigma) and activated using 100 ml of ethyl(dimethylamino- propyl) carbodiimide (EDC) (100 mg/ml) with immediate addition of 100 ml of N-hydroxy succinamide NHS (100 mg/ml) (both from Pierce Biotechnology) and the reaction was carried out for 20 minutes at room temperature.Afterwards, beads were washed with MES-T buffer using centrifugation at 600 G for 8 minutes on 3-mm Transwell inserts (Life Technologies) assembled on top of 15-ml centrifuge tubes. Washing procedure was repeated twice in order to remove excess of unreacted EDC and NHS. Afterwards, the beads were aspirated into a 1.5-ml Eppendrof tube using 150 ml of DMSO/MES-T (50%/50% mixture) and mixed with 30 mg of ganglioside, respective to the selected bead number. Reaction was allowed to go for 24 hours on a planetary shaker at 600 rpm and 4uC, and then quenched by adding 600 ml of 50 mM glycine buffer pH 7.3. The resultant mixture was incubated for 15 hours at 4uC and 600 rpm shaking. Upon completion, the final gangliosides-conjugated beads were washed in cold PBS through centrifugation at 600 G for 8 minutes using 3-mm Transwell inserts assembled on tops of 15-ml centrifuge tubes and collected in PBS containing 2% Grade V BSA (bovine serum albumin) and 0.1% sodium azide (Sigma). Finally, 10% v/v of glycerol was added, and the bead suspension was stored in liquid nitrogen. The bead number was calculated using a hemocytometer. Testing of a standard anti-gangliosides rabbit serum Standard rabbit sera against the gangliosides GM1, GM2, GA1 and GD1b were obtained from Matreya LLC. Each serum was serially diluted two-fold starting from 1:100 to 1:3276800. Sera dilution was made in 70/30 mixture of PBS/DMSO. 100-ml portions of the diluted anti-ganglioside rabbit sera were incubated in duplicates with corresponding ganglioside-conjugated beads in a 96- well Bioplex filter plates (Bio-Rad) at 4uC for 150 minutes at 600 rpm shaking. Afterwards, the wells were washed twice with 85 ml of PBST/NaN3 for 2 minutes on a planetary shaker at 600 rpm at room temperature filtering the plates on a vacuum manifold after each wash. Afterwards, 85 ml (1 mg/ml in 70/30 PBS/DMSO) of biotinylated anti-rabbit IgG(H+L)Fab2:biotin (Jackson Immunoresearch) was applied as a detecting antibody, and the plate was incubated at 4uC for 40 minutes at 600 rpm. Again, the wells were washed twice with PBST/NaN3 as described above, and 85 ml of 4 mg/ml of streptavidin-phycoerythrin (SA-PE) fluorescent conjugate (Leinco Technologies, Inc.) was introduced PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42681