Autoimmune, Infectious Disease Multiplexing Tool Figure 5. Testing multiplexing capacity of combined ganglioside-influenza bead array. Luminex beads coated with various payloads of Brisbane H1N1 and California H1N1 hemagglutinins were tested, and beads with the payload 0.5 mg (encircled in red) was selected for the combined ganglioside-influenza bead array (a, b). As a proof of principle, ganglioside-influenza bead array was tested with sera from two donors vaccinated against seasonal influenza in 2009 (c) and against pandemic California H1N1 in 2010 (d). Increase of influenza-specific antibodies post vaccination is apparent. More details in the text. (Note: Errors were too small to include in the panels a and b). Anti-Brisbane H1N1 and anti-California H1N1 sheep sera (NIBSC, UK) were used at dilution 1: 50000 as a positive control for detecting influenza-specific antibodies by hemagglutinin-coated beads. Anti- ganglioside rabbit sera (Matreya LLC) were used as positive controls for detecting ganglioside-specific antibodies at the following dilutions: anti-GM1 - 1:6400, anti-GM2 - 1:800, anti-GA1 – 1:25000, anti-GD1B – 1:400. doi:10.1371/journal.pone.0042681.g005 tin. All sera dilutions and detecting antibody solutions were made in 70%/30% PBS/DMSO mixture. Similarly, ELISA assays were carried out by coating the Immulon 4HBX strips with 100 ml per well of ganglioside solutions at 5 mg/ml in duplicates, in 70/30 mixture of PBS/DMSO overnight. Next day, the plates were blocked with 200 ml/well of 2% BSA for 2 hours. Then plates were incubated with 50 ml of CTB (2 mg/ml) (Sigma Aldrich) or 50 ml of AS (15 mg/ml) (Sigma Aldrich) at 4uC for 1 hour at 500 rpm shaking. Then plates were washed twice with PBST and respective anti-rabbit ganglioside specific sera were introduced. After incubation for 2 hours, the plates were washed twice with PBST and anti-rabbit IgG(H+L)Fab2:biotin detecting antibody was added at 2 mg/ml, 85 ml/well, and incubated for 1 hour. Next, the plates were washed with PBST thrice and incubated with 85 ml/well of 4 mg/ml of SA-PE for 1 hour. Finally, the plates were washed with PBST four times and read on BioTek plate reader using phycoerythrin filter setting. All the experiments were carried out in duplicates. Buhlmann GM1 ELISA – a golden standard assay Buhlmann ELISA kit for GM1-specific antibodies (IgG and IgM) was used as a golden standard assay in this study. The kit (EK-GM1-GM, Buhlmann Laboratories AG, Switzerland) con- tains a microtiter plate coated with GM1 antigen (the formulation of which is not specified in the product manual), and blocked by the manufacturer. The kit also contains calibrator and control (standard) solutions of GM1-specific sera, detecting antibodies tagged with horseradish peroxidase (HRP), TMB peroxidase substrate, a stop solution and washing and dilution buffers. The ELISA is based on a regular colorimetric technique using peroxidase driven oxidation of the TMB substrate into a blue chromophore and turning it into a yellow chromophore using an acidic stop solution. The optical density of the yellow chromo- phore is read in the plate reader at 450 nm. The whole procedure of applying calibrator, control and tested samples, washings, application of the detecting antibodies, reading takes ,4.5 hours [38]. PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e42681