33 research outputs found

    Signals.

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    <p>Bar chart displaying the fluorescence signal obtained from binding of polyclonal anti-HSA to 15-mer peptides with 14 residue overlap (average signal from 5 copies of each peptide). <b>A:</b> peptides from HSA, <b>B:</b> peptides from BSA and <b>C:</b> peptides from RSA. The peptides are numbered on the x-axis according to the position of their n-terminal residue in the protein sequence. The y-axis denotes the average intensity of the signal (AU) after background subtraction.</p

    All data: Binding of anti-HSA antibody to Array 2 (Identical to/Repeat of Array 1)

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    Datafile from peptide microarray measuring Human Serum Albumin(HSA) antibody binding to peptides from HSA and variations of these peptides

    All data: Binding of anti-HSA antibody to Array 1

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    Datafile from peptide microarray measuring Human Serum Albumin(HSA) antibody binding to peptides from HSA and variations of these peptides

    Identification and Mapping of Linear Antibody Epitopes in Human Serum Albumin Using High-Density Peptide Arrays

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    <div><p>We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm<sup>2</sup>. Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.</p></div

    Image of array.

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    <p>A small section of the peptide array used for identification and fine specificity mapping of HSA epitopes: The section shows approximately 300 of the total 220.428 peptide fields in this array. The peptides were synthesized in predefined, addressable fields generated by 2×2 mirrors on the DMD each measuring 10×10 µm resulting in peptide fields with the size 20×20 of µm. The peptide fields were spaced by 10 µm wide empty zones. Binding of polyclonal rabbit anti-HSA antibody to the fields was recorded by fluorescence microscopy after incubation with Cy3-conjugated goat anti-rabbit IgG.</p

    Example of Tukey’s HSD.

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    <p>The figure shows two examples example of epitope sequences identified using ANOVA followed by Tukey’s HSD post-hoc analysis. The leftmost column shows overlapping 15-mer peptides from HSA from position 510–527 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068902#pone-0068902-g003" target="_blank">figure 3A</a> and from position 309 to 333 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068902#pone-0068902-g003" target="_blank">figure 3b</a>. The rightmost column highlights the amino acids identified as being important for antibody binding (dashes indicate non-important positions). The important amino acids were determined on the p<0.01 level in the HSD post-hoc analysis.</p

    Example of mean Rq-ratio.

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    <p>The figure shows two examples using the Rq-values. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068902#pone-0068902-g004" target="_blank">Figure 4a</a> shows residue 510–527 in the HSA sequence (top row) a sequence containing the epitope important residues LEVDETYV identified by Tukey’s HSD. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068902#pone-0068902-g004" target="_blank">Figure 4b</a> shows residue 317 to residue 338 containing the epitope important residues DEMPADLP-LAADFVESKD. The column below each HSA residue lists the Rq-values calculated in each of the 15 overlapping peptides in which the residue is represented. Rq values from peptides with no positions identified by Tukey’s HSD test are set as non significant (NS) in the figure. The color indicates the size of the Rq value; darker color indicates higher Rq values.</p

    Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction-1

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    <p><b>Copyright information:</b></p><p>Taken from "Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction"</p><p>http://www.biomedcentral.com/1471-2105/8/424</p><p>BMC Bioinformatics 2007;8():424-424.</p><p>Published online 31 Oct 2007</p><p>PMCID:PMC2194739.</p><p></p>ng NetCTL-1.2 is compared to the rank assigned when using the test method (EpiJen, MAPPP, MHC-pathway, or WAPP). The height of the bars indicates how often, respectively, NetCTL or the test method ranks the epitope highest. The HIV dataset has been used for the analysis. When comparing NetCTL-1.2 to either of the test methods, only predictions for supertypes that the test method covers are included. The HIVdataset has been used for the analysis. ** The difference is significant at P < 0.01. * The difference is significant at P < 0.05

    Flow diagram of the cell analysis.

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    <p>At day 1, some PBMC's were use directly for an <i>ex vivo</i> analysis of pp65 specific T cells. The remaining PBMC's were split in adherent and non-adherent cells. The non-adherent cells comprising the T cells were expanded on a pp65 peptide mixture. The peptide mixture was added overnight, washed away the next day, and the T cells were propagated on IL2 until day 7. At the same time, the adherent cells were cultured for 7 days in the presence of IL4 and GM-CSF to mature the into DC. At day 7, both the T cells and the DC's were harvested. The DC's were pulsed with the pp65 peptide mixture and added to the T cells (at the ratio 1∶10) during a 4 h ICS assay.</p
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