Buhlmann Labs also supply separately the GM1-specific human sera standard, which makes it a convenient sample for compar- ative sensitivity tests. Evaluation of epitope specificity using CTB and AS blockers in Buhlman GM1 ELISA kit ELISA strip coated with GM1-ganglioside as provided in the kit was pre-incubated with 50 ml of CTB (2 mg/ml) and 50 ml of AS (15 mg/ml). The rest of the procedure was performed following the protocol supplied in Buhlmann ELISA kit. All the measurements were carried out in duplicates. Evaluation of reactivity For cross reactivity evaluation, each type of ganglioside- conjugated beads was incubated with 100 ml of particular dilution of the corresponding anti-ganglioside sera (1:6400 of anti-GM1, 1:50000 of anti-GA1, 1:1600 of anti-GM2 and 1:800 of anti- GD1b sera) in duplicates in a 96-well plate at 4uC for 150 minutes at 600 rpm. The dilutions were chosen to obtain comparable reporter signals for different sera, based on the previous sera titrations. The sera and anti-rabbit IgG(H+L)Fab2:biotin detecting antibody were diluted in 70%/30% PBS/DMSO mixture. Bioplex measurement protocol was used as described in the earlier sections. Cross-reactivity was estimated by comparing the reporter signal from the beads coated with the ganglioside related to the tested serum and the signals from the beads coated with other gangliosides. Comparing concentration sensitivity of ganglioside- conjugated bead array and ELISA Concentration sensitivity measurements were done using either ganglioside-specific rabbit sera, or Buhlmann GM1 sera standard, in parallel for both BioPlex and ELISA measurements. Two-fold serial dilution starting from 1:100 was used. Sera samples were diluted in 70%/30% PBS/DMSO mixture. Ganglioside conju- gated beads were incubated with 100 ml of serially diluted sera for 2.5 hours. After bead washing, 85 ml of goat anti-rabbit IgG(H+L)Fab2:biotin or donkey anti-human IgG(H+L)Fab2:biotin detecting antibody were applied as a detecting antibody, and SA- PE used as a fluorescent tag. BioPlex measurement procedure was used as described in the earlier sections. In-house ELISA was carried out by coating the Immulon 4HBX strips with 100 ml of 5 mg/ml ganglioside solution in 70/30 mixture of PBS/DMSO per well in duplicates, overnight. Next day, the plates were blocked with 200 ml/well of 2% BSA for 2 hours. Then the plates were washed twice with PBST and respective rabbit anti-ganglioside sera were introduced, serially diluted starting with dilution 1:100. After incubation for 2 hours, the plates were washed with PBST twice, and anti-rabbit IgG(H+L)Fab2:biotin antibody was added at 2 mg/ml, 85 ml/well, and the plates incubated for 1 hour. Next, the plates were washed with PBST thrice and incubated for 1 hour with 85 ml/well of of SA-PE at 4 mg/ml. 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 ELISA using the internal GM1 sera standard as a sample was carried out according to the Buhlmann kit protocol. The GM1 specific sera standard was applied serially diluted, starting with dilution 1:100., in duplicates. Autoimmune, Infectious Disease Multiplexing Tool Fabrication of beads coated with influenza hemagglutinin Fluorescent carboxylated non-magnetic beads (Luminex) were used, #42 for California H1 recombinant hemagglutinin, and #12 for Brisbane H1 recombinant hemagglutinin (both from Protein Sciences). First, 50 ml of bead suspension (from stock 1.25*107 beads/ml) was diluted in 270 ml of 100 mM pH 3.5 MES-T buffer and activated using 40 ml of ethyl(dimethylaminopropyl) carbodii- mide (EDC; 100 mg/ml, Pierce Biotechnology) and 40 ml N- hydroxy succinamide (NHS, 100 mg/ml, Pierce Biotechnology) for 20 minutes at room temperature. Afterwards, beads were washed with MES-T buffer through centrifugation at 600 G for 8 minutes, using 3-mm Transwell inserts as described above. Washing procedure was repeated twice in order to remove excess of unreacted EDC or NHS. Then the beads were aspirated into a 1.5-ml Eppendrof tube using 150 ml of MES-T and an aliquot of 10 mg, 0.5 mg, 0.3 mg or 0.1 mg of hemagglutinin was admixed. The reaction was allowed to go for 24 hours at 4uC on a planetary shaker at 600 rpm and then quenched by adding 600 ml of 50 mM glycine buffer pH 7.3 (Sigma). The mixture was incubated for 15 hours at 4uC on a planetary shaker at 600 rpm. Upon completion of conjugation reaction, the final gangliosides-conju- gated beads were washed in cold PBS through centrifugation at 600 G for 8 minutes using 3-mm Transwell inserts as described above, and collected in PBS containing 2% BSA (bovine serum albumin) and 0.1% sodium azide. Finally, 10% v/v of glycerol (Sigma) was added, and the bead suspensions were stored in liquid nitrogen. The bead number was calculated using a hemocytometer. Demonstration of multiplexing capacity of ganglioside and influenza bead array Human pre- and post-vaccinated sera collected from donors vaccinated against seasonal influenza in 2009 and against pandemic California H1N1 virus in 2010 were tested at dilution 1:2000 in the 70%/30% PBS/DMSO mixture. Prior to incuba- tion with sera samples, the beads were combined in a pentaplex array containing GM1-, GM2-, GA1-, GD1b-conjugated beads, and Brisbane H1 HA coated beads, or California H1 HA coated beads. Human sera samples were incubated with bead arrays for 150 minutes at 4uC at 600 rpm. After that, the plate was washed twice with PBST/NaN3 and then 85 ml (2 mg/ml in 70/30 PBS/ DMSO mixture) of anti-human donkey IgG(H+L) Fab2:biotin was applied as a detecting antibody. The remaining washing proce- dure, application of SA-PE and reading on Bioplex were similar to the BioPlex protocol described in the earlier sections. 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. Supporting Information Figure S1 Selection of optimal secondary detecting antibody. Donkey IgG(H+L)Fab2:biotin from Jackson Immu- noresearch was selected for detection of ganglioside-specific IgG and for most of the BioPlex experiments. MFI = Mean Fluores- cence Intensity. (TIF) PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e42681