Autoimmune, Infectious Disease Multiplexing Tool Figure 2. Evaluating bead epitope specificity using CTB and AS epitope blockers. CTB has ability to bind 5 GM1 molecules (a) compared to alpha-synuclein that can bind only one GM1 molecule (b). CTB showed ,90% bead blocking with anti-GM1 rabbit serum compared to ,15% with other anti-ganglioside rabbit sera while synuclein exhibits ,60–90% blocking with all anti-ganglioside sera (c). Screening human sera, 20%–40% blocking was seen with CTB compared to 70%–80% using AS (d). The radical R circled in dashed red signifies the presence of sialic acid groups (one in GK1 and GM2, two in GD1b and none in GA1). doi:10.1371/journal.pone.0042681.g002 one adjacent to ceramide moiety, the epitopes would not be readily available for ganglioside-specific antibodies. Having the ganglioside bead arrays fabricated and tested with anti-ganglioside sera, we performed selection of the optimal secondary detecting antibody. For this purpose, a standard human anti-GM1 serum from Buhlmann ELISA kit (EK-GM1-GM, Buhlmann Laboratories AG, Switzerland) was used as the test sample. Various secondary antibodies from different vendors were applied such as goat anti-human IgG:biotin (Southern Biotech), goat anti-human (IgG+IgM)(H+L):biotin, donkey anti-human IgG(H+L)Fab2:biotin and goat anti-human IgG(Fc):biotin (all three from Jackson Immunoresearch). We found that donkey anti-human IgG(H+L) Fab2:biotin provided a considerably higher reporter signal compared to the other secondary antibodies (Figure S1), and this antibody was used in the subsequent Bioplex measurements of ganglioside-specific IgG. Epitope specificity and cross reactivity determination of ganglioside high throughput multiplexing tool It is well known that the cholera toxin beta subunit (CTB) conjugates specifically and strongly with the carbohydrate/sialic acid epitope of the GM1-ganglioside but has considerably lower affinity towards other gangliosides [31], [32]. Another potential blocker, alpha-synuclein (AS) binds specifically to carbohydrate and sialic acid residues of GM1-ganglioside [33] (Figure 2a and 2b). Although the binding capacity of alpha-synuclein to other gangliosides is not well characterized. We carried out the epitope specificity tests using both CTB and AS as ganglioside epitope blockers. The ganglioside-conjugated beads were individually pre- incubated with each blocker, and after that rabbit anti-ganglioside sera were added. As expected, CTB was able to block almost 100% of the signal from the GM1 coated beads, and showed much lower effect on beads coated with other gangliosides (Figure 2c). Alpha-synuclein showed affinity to all ganglioside-conjugated beads blocking from 60% to 90% of fluorescent signal (Figure 2c). However, in the measurements of human sera, only 20% to 40% blocking with CTB was observed while AS showed 70% to 80% blocking (Figure 2d). This result assumes that human sera may contain GM1-reacting antibodies specific to the epitopes other than the known carbohydrate/sialic acid epitope, probably including a certain part of the ceramide moiety. Similarly, effects of blockers were assessed by performing ganglioside-specific ELISA for GM1, GM2, GA1 and GD1b ganglioside (Figure S2). The ELISA assay demonstrated blocking effect of CTB with GM1 ganglioside only, and no blocking effect of AS with each of the four gangliosides. Additionally, we tested the effect of blockers using commercially available Buhlmann anti- human GM1 sera standard. Buhlmann ELISA has been designed to quantitatively determine IgG/IgM isotypes of autoantibodies directed against GM1 in human serum applying individual IgG- and IgM-specific conjugates. Interestingly, using internal Buhl- mann anti-GM1 sera standard in the ELISA experiment, we did not see any blocking effect of CTB or AS (Figure S3), Blockers assessment with other ganglioside-specific human sera standards such as anti-GA1, anti-GM2 or anti-GD1b were not done in this study due to their commercial unavailability. We assume that lack PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42681