of blocking capacity in the ELISA experiments could be explained by a different orientation of gangliosides bound on the ELISA plates compared to the gangliosides conjugated to the bead array, where they are oriented in a pendant-like projection readily available for binding with the blockers, while in the ELISA plate this favorable orientation may not be possible. These results demonstrate the unique ability of multiplexing tool over ELISA to demonstrate epitope specificity of the ganglioside-reacting anti- bodies using epitope blockers such as CTB and AS. In order to further characterize specificity of the ganglioside HTM tool, cross reactivity of ganglioside-conjugated beads were tested using commercially available anti-ganglioside rabbit sera. A typical ganglioside molecule contains a carbohydrate sequence with a varying number of sialic acid residues attached to the carbohydrate backbone [31],[33]. The structural similarities among various gangliosides may result in cross reactivity of ganglioside antibodies to the beads coated with different gangli- osides which may affect the assay results. ELISA and TLC (thin layer chromatography) have been used by others to assess the cross reactivity of ganglioside specific antibodies [34], [35], but the new ganglioside HTM tool needed to be characterized independently. A significant cross reactivity for anti-GM2 and anti-GD1b sera compared to anti-GM1 and anti-asialo-GM1 sera was found (Figure 3a, 3b, 3c, 3d). GM1-, asialo-GM1- and GD1b- gangliosides have common terminal sugar sequence (Figure 3e, dotted red square), hence beads coated with GM1-ganglioside and asialo-GM1 ganglioside exhibit a significant cross reactivity to the anti-GD1b sera. In the same time, general prediction of the sera cross reactivity can be complicated by its dependence on the particular epitope spectra of the antibodies included in the anti- sera. For example, cross reactive antigen-antisera relations for GM1 and GM2 gangliosides were found asymmetrical; for example anti-GM1 serum did not react with GM2 bead while anti-GM2 serum did react with GM1 bead. Hypothetically, the terminal galactose of the GM1 ganglioside was a critical part of the major reactivity epitope for the anti-GM1; therefore lack of the terminal galactose in the GM2 banned interaction of anti-GM1 serum with GM2 bead (Figure 3e). On the other hand, the major epitope of the anti-GM2 can include Gal-NAc-Gal-Sialic Acid construct common for all four gangliosides used in the array. The cross reactivity of GM1-beads towards anti-asialo-GM1 serum was very insignificant, which reflected the importance of sialic moiety in shaping up the GM1 epitopes. Also, lack of cross reactivity of GD1b-ganglioside beads towards anti-GM1 and anti-asialo-GM1 sera can be attributed to the extra sialic acid in the GD1b structure, which may prevent binding of anti-GM1 and anti- asialo-GM1 antibodies due to the steric effect. Comparing concentration sensitivity of the multiplexing tool and ELISA As stated earlier, ELISA is widely used for the detection of ganglioside antibodies, but concentration sensitivity of ELISA assay is limited. We assumed that ganglioside-conjugated beads may exhibit better concentration sensitivity than ELISA due to the more appropriate orientation and better exposure of the gangli- oside epitopes. We compared the concentration sensitivity of ganglioside-conjugated Luminex beads and the traditionally used ganglioside-specific ELISA for all four mentioned gangliosides GM1, GM2, GA1 and GD1b. Experiments were carried out with the commercially available standard rabbit anti-ganglioside serum as a tested sample, using two-fold serial dilution from 1:100. For both the Bioplex and ELISA experiments, biotinylated donkey anti-rabbit IgG(H+L) Fab2:biotin was used for measuring IgG fraction, with SA-PE as a fluorescent tag. The concentration Autoimmune, Infectious Disease Multiplexing Tool sensitivity of the ganglioside-conjugated beads in the BioPlex experiment was found 10–100 times better than the ELISA assay for IgG detection, (Figure 4a, 4b, 4c, 4d). Similar comparison of the concentration sensitivity was performed using internal anti- GM1 standard. In this case, the ELISA assay used the original Buhlmann protocol and colorimetric registration. The concentra- tion sensitivity of the ganglioside-conjugated beads in this test was found ,50 times better than in the ELISA assay for IgG and ,100 times better for IgM (Figure S3). Demonstration of multiplexing capacity for infectious- disease specific antibodies In our previous experiments with influenza bead arrays, the concentration sensitivity of influenza coated beads was found very high, and the dilutions normally used in measuring influenza- positive sera were from 1:50000 to 1:500000 (data not shown). Using such dilutions was not appropriate for simultaneous detection of ganglioside-specific autoantibodies in human sera, since the auto-antibodies are scarce compared to the antibodies of the influenza vaccination response. In order to allow simultaneous measurements of influenza- and ganglioside-specific antibodies with the same bead array, the sensitivity of the influenza beads was intentionally reduced by decreasing the payload of influenza hemagglutinins on the beads. This approach allowed screening of sera at dilutions 1:100–1:2000 (Figures 5a and 5b). As a proof of the principle, human sera from donors immunized with vaccines containing Brisbane H1N1 and California H1N1 components were tested at dilution 1:2000 using pentaplex bead array containing various ganglioside and influenza beads. As positive controls, anti-Brisbane H1N1 and anti-California H1N1 sheep sera were used for hemagglutinin-coated beads, and ganglioside- specific rabbit sera for ganglisoide-coated beads. As expected, the multiplex tool successfully screened both influenza and ganglioside antibodies in a single sample volume (Figure 5c and 5d). In addition to assessing the levels of ganglioside-specific antibodies, those multiplexing measurements showed a 2-fold increase in influenza-specific antibodies [36], [37] post vaccination, but no change in the ganglioside-specific antibodies. The experiment therefore demonstrated the capacity of the multiplex tool to assess both infectious disease and autoimmune responses in a single run and the fact that influenza immunization normally does not cause autoimmunity. Discussion Concluding, we report development of a novel high throughput multiplexing tool based on the bead array technology, which provides simultaneous measurements of autoimmune antibodies specific to various neuronal gangliosides together with antibodies to an infectious disease (influenza). The bead array was fabricated using modified carbodiimide chemistry in an aqueous/organic media, where linking of the gangliosides occurred via the secondary amine group adjacent to the ceramide moiety. The ganglioside-influenza bead array exhibited excellent detection limits offering 10–100 times better concentration sensitivity to ganglioside-specific antibodies than the ELISA assay, including the golden standard, Buhlmann GM1 ELISA kit. Moreover, the array demonstrated excellent reactivity towards ganglioside blockers, CTB and AS, which proved epitope specificity of the detected antibodies. Combining ganglioside- and influenza-specific ele- ments in one instrument provided an exclusive capability to measure immune response to infectious disease or vaccination and their possible autoimmune effects in a simultaneous assay. Therefore, potential application of such type of a multiplexing PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42681