Testing multiplexing capacity of combined ganglioside-influenza bead array.

<p>Luminex beads coated with various payloads of Brisbane H1N1 and California H1N1 hemagglutinins were tested, and beads with the payload 0.5 µg (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.</p

Cross-reactivity of ganglioside antibodies.

<p>GM1- and GA1-conjugated beads showed considerable cross reactivity with anti-GD1b sera since these gangliosides have common terminal sugar sequence <b>(panels b, c; dotted red square in panel e)</b>. GM1 beads displayed cross-reactivity with anti-GM2 serum due to the shared epitope groups in the inner part of the GM1- and GM2-gangliosides <b>(panel d; dotted black square in panel e)</b>. Cross-reactivity was estimated 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.</p

Evaluating bead epitope specificity using CTB and AS epitope blockers.

<p>CTB has ability to bind 5 GM1 molecules (<b>a</b>) compared to alpha-synuclein that can bind only one GM1 molecule (<b>b</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 (<b>c</b>). Screening human sera, 20%–40% blocking was seen with CTB compared to 70%–80% using AS (<b>d</b>). 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).</p

Concentration sensitivity to ganglioside-specific sera of BioPlex bead array, compared to ELISA.

<p>Ganglioside-conjugated Luminex bead exhibited approximately 100 times better sensitivity to the sera specific to GM1, GM2 and GG1b gangliosides (a, b, d), and about 10 times better sensitivity to the serum specific to GA1 (c). Both BioPlex and ELISA assays used the same detecting antibody, anti-rabbit IgG(H+L)Fab2:biotin at 2 ug/ml, and SA-PE fluorescent tag at 4 ug/ml. Dashed circles show approximate sensitivity thresholds for BioPlex (blue) and ELISA (red).</p

Nanoceria Facilitates the Synthesis of Poly(<i>o</i>‑phenylenediamine) with pH-Tunable Morphology, Conductivity, and Photoluminescent Properties

Poly­(<i>ortho</i>-phenylenediamine) synthesis enabled by the catalytic oxidase-like activity of nanoceria was accomplished for applications in electronics, medicine, and biotechnology. The polymer shows unique morphology, conductivity, and photoluminescence based on pH of the solution during synthesis. The various poly­(<i>ortho</i>-phenylenediamine) preparations were characterized by UV–visible spectroscopy, scanning electron microscopy, fluorescence spectroscopy, fluorescence microscopy, high-pressure liquid chromatography, and cyclic voltammetry. Poly­(<i>ortho</i>-phenylenediamine) synthesized at pH 1.0 by nanoceria was selected to be extensively studied on the basis of the fast synthetic kinetics and the resulting conductive and photoluminescent properties for various applications

Schematic representation of Control ELISA to check for false positive effects in the fADI neutralization assay.

<p>Virus is added to ELISA plates instead of using target cells to check for any false positive readings. Addition of sera (upper arm) or no sera (lower arm) should produce similar reporter signals (fluorescence) indicating the absence or low level of <i>competition</i> of virus-specific labeling antibody with antibodies in test serum. The plates are read on the Synergy HT plate reader (BioTek) after applying the secondary detecting antibody and the fluorescent tag SA-PE. More details are in the text.</p

Correlation between fADI and PRNT methods for Yellow Fever vaccine virus.

<p>fADI₅₀ were calculated from the Virus Attachment Indexes using a mathematical approximation model, more details in the text. The fADI titers for all the pre-vaccination sera samples were found very low. fADI displayed a considerable donor-to-donor variation in the capacity of post-vaccination donor sera to block attachment of vaccine strain 17DD of Yellow Fever virus to Vero cells (a). PRNT₅₀ data were obtained for the same sera samples independently by a commercial vendor (b). Control ELISA did not reveal a reduction of the reporter fluorescence signal, thus showing zero competition between the sera antibodies and the virus specific labeling antibodies used in the assay (c). A considerable positive correlation between the fADI and PRNT data for post-vaccination sera samples was found (d).</p

Correlation between fADI and MN methods for Polio I, II and III.

<p>fADI₅₀ were calculated from the Virus Attachment Indexes using a mathematical approximation model (see text for details). fADI showed a significant correlation of virus attachment blocking for polioviruses of serotypes I, II and III with the data of microneutralization assay obtained independently at CBER/FDA using the WHO miconeutralization protocol.</p

fADI screening of pre- and post-vaccinated serum samples from donors immunized with IPOL^® vaccine.

<p>The screening was performed in the single dilution mode rather than using sera titration. Donor-to-donor variation in the capacity of donor sera to block virus attachment was observed for all three poliovirus serotypes (A, B and C). The control ELISA tests (D, E and F) did not display significant reduction of the reporter fluorescence signal, thus showing very low or zero competition between test serum antibodies and virus-specific labeling antibodies used in the assay.</p

Proof of principle study for the fADI assay against different classes of viruses.

<p>fADI displayed a significant sera concentration dependent blocking of virus attachment for polioviruses of serotyopes I, II and III (A), Dengue I, II, III and IV (C), Yellow Fever (E) and influenza California H1N1viruses (G) to Vero cells. The sera used in the demo experiments were standard hyper-immune sera specific to the corresponding viruses. Control ELISA (B, D, F and H) tests with those standard sera did not display any significant reduction in fluorescent signal thus showing very low or no competition between the sera antibodies and virus specific labeling antibodies used in the assay.</p