Results Synthesis and characterization of ganglioside-conjugated beads Ganglioside-conjugated bead arrays were fabricated using carbodiimide chemistry. A typical ganglioside molecule does not contain primary amine groups, which are typically used for conjugation with carboxyl groups, including those on the surface of Luminex beads which are used in the current study. However, we hypothesized that the conjugation of gangliosides could be achieved via the secondary amine groups adjacent to the ceramide moiety in ganglioside structure. Conjugation over another secondary amine group situated in the sialic acid residue was considered less feasible due to the possible steric hindrance. The gangliosides selected for coating the beads, GA1, GM1, GM2 and GD1b are known for clinical significance of the auto-antibodies towards these antigens in various neuropathic disorders [6–9]. The gangliosides were conjugated to the surface of carboxylated fluorescent Luminex beads, their code numbers 45, 27, 25 and 14 respectively, using a modified carbodiimide chemistry protocol (Figure 1a; details of the protocol below). Manipulations with gangliosides in aqueous solutions create certain problems. Gangliosides can be easily dissolved in organic solvents such as DMSO (dimethyl sulfoxide), but develop micelles in watery buffers. Also, gangliosides easily lose their antigenic properties when stored in buffers at room temperature, which indicates conformational changes or chemical instability of the molecules. To avoid these problems, conjugation to the beads was carried out at 4uC in 1:1 aqueous/organic mixture of MES-T buffer (0.05% v/v Tween 20 in 50 mM MES, pH 3.5) and DMSO. Upon completion of the conjugation reaction, the final Autoimmune, Infectious Disease Multiplexing Tool ganglioside conjugated beads were washed in ice cold PBS and collected in PBS containing 2% w/v BSA (bovine serum albumin) and 0.1% w/v sodium azide. Successful conjugation of ganglio- sides to Luminex beads was confirmed by testing them with commercially available rabbit anti-ganglioside sera. Ganglioside- conjugated beads were able to capture the anti-ganglioside antibodies and display a dilution dependent fluorescent response (Figure 1b). Despite considerable hydrophobicity of the whole ganglioside molecules, their epitopes consist of hydrophilic carbohydrates and sialic groups. In order to reconcile the hydrophilic and hydrophobic propensities of the ganglioside components and to maintain proper conformations of the molecules on the Luminex beads, the tested sera and biotinylated detecting antibodies were diluted in aqueous/organic mixture of PBS/DMSO (70%/30% v/v). The streptavidin-phycoerythrin fluorescent tag (SA-PE) dissolved in 1% BSA was applied at the end of the assay. Coupling of gangliosides to the beads via secondary amide bond was assessed by the bond hydrolysis driven by ceramidase and lipase enzymes which are able to hydrolyze both primary and secondary amide bonds (Figure 1c) [26–28]. As expected, Bioplex experiments showed lack of the reporter fluorescent signal after treatment of beads with ceramidase or lipase (Figure 1d). Successful coupling of the asialo-GM1 ganglioside to the beads further confirmed our hypothesis that conjugation took place via the secondary amine group adjacent to ceramide moiety, since asialo-GM1 ganglioside does not possess a sialic acid and the sialic secondary amine group. Another consideration in favor of the conjugation of gangliosides via ceramide-adjacent nitrogen is a sheer fact that the carbohydrate and sialic acid moieties serve as key ganglioside epitopes [29], [30]. If the conjugation would have occurred via nitrogen other than the Figure 1. Synthesis and characterization of ganglioside bead array. A scheme of conjugation of various gangliosides on Bioplex beads via modified EDC/NHS chemistry (a). Dilution-dependent fluorescent response displayed by ganglioside conjugated beads testing various anti- ganglioside rabbit sera (b). Coupling of gangliosides to beads occurred via secondary amine next to ceramide moiety (c). Ceramidase and lipase treatment of beads negate the reporter fluorescent signal (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.g001 PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42681