Autoimmune, Infectious Disease Multiplexing Tool Figure 4. Concentration sensitivity to ganglioside-specific sera of BioPlex bead array, compared to ELISA. Ganglioside-conjugated Luminex bead exhibited approximately 100 times better sensitivity to the sera specific to GM1, GM2 and GG1b gangliosides (a, b, d), and about 10 times better sensitivity to the serum specific to GA1 (c). Both BioPlex and ELISA assays used the same detecting antibody, anti-rabbit IgG(H+L)Fab2:biotin at 2 ug/ml, and SA-PE fluorescent tag at 4 ug/ml. Dashed circles show approximate sensitivity thresholds for BioPlex (blue) and ELISA (red). doi:10.1371/journal.pone.0042681.g004 into each well. After incubation for 40 minutes at 4uC, the plate was washed twice with PBST/NaN3 as described above, and once with PBS/NaN3 containing no detergent. Then 85 ml of PBS/NaN3 was added to each well, and the plate was shaken for 5 minutes at 1300 rpm at room temperature to avoid settling of the beads. Finally, another 55 ml of PBS/NaN3 was added to each well, and the plate was shaken at 600 rpm for one minute. After that, the plate was read on Bioplex-100 bead array reader (Bio-Rad). As a negative (mock-) control, 100 ml mixture of PBS/DMSO was used. Similar protocols were used for measuring or detecting ganglioside antibodies in all assays, including studying human donor sera. All the experiments were carried out in duplicates. BioPlex experiment after ceramidase/lipase treatment Individual beads were incubated in 10 mg/ml ceramidase or lipase (Sigma Aldrich) at 4uC overnight at 600 rpm shaking. Then the treated beads were incubated in duplicates with 100 ml of anti- ganglioside sera at 4uC for 150 minutes at 600 rpm. Remaining procedure was followed as described in the above section. Selection of optimal detecting antibodies Four different types of detecting antibodies were tested using gangliosides-conjugated beads and human anti-GM1 gangliosides serum from Buhlmann GM1 ELISA kit. For IgG, goat anti- human IgG:biotin (Southern Biotechnology), goat anti-human IgG(H+L)Fab2:biotin, goat anti-human (IgG+IgM)(H+L)Fab2 :biotin and donkey anti-human IgG(H+L)Fab2:biotin (all three from Jackson Immunoresearch) were screened. For IgM, goat anti- human IgM:biotin (Southern Biotechnology), Goat anti-human IgM(H+L)Fab2:biotin, goat anti-human (IgG+IgM)(H+L)Fab2:bio- tin and donkey anti-human IgM(H+L)Fab2:biotin (all three from Jackson Immunoresearch). Bioplex experiment was set as described above with only difference of using human anti-GM1 ganglioside sera instead of the rabbit sera, and applying human specific rather than rabbit specific detecting antibodies. Sera and detecting antibody were diluted in 70%/30% PBS/DMSO mixture. All antibodies were tested in duplicates. Evaluation of epitope specificity employing CTB and AS ganglioside blockers – Bioplex and ELISA assays using the same components To test epitope specificity in the BioPlex experiment, the standard Bioplex protocol described above was followed, except that the individual ganglioside conjugated beads were pre- incubated with 50 ml of CTB (2 mg/ml) (Sigma Aldrich) or 50 ml of AS (15 mg/ml) (Sigma Aldrich) in duplicates in a 96-well plate at 4uC for 1 hour at 500 rpm shaking. Then 50 ml of anti-rabbit ganglioside sera were introduced into respective wells (1:6400 of anti-GM1, 1:50000 of anti-GA1, 1:1600 of anti-GM2 and 1:800 of anti-GD1b sera). After performing the whole procedure, the plate was read in the Bioplex reader. A similar experiment was carried out employing human sera at dilution of 1:2000, but donkey anti- human IgG(H+L)Fab2:biotin (Jackson Immunoresearch) was used as a detecting antibody instead of anti-rabbit IgG(H+L)Fab2:bio- PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42681