Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.

<p>Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.</p

Enrichment of phage clones predicted to display authentic NadA fragments on their surface after selection with a serum pool from volunteers immunized with the Bexero vaccine.

<p>Frequency values reported in the vertical axis in panels A–C refer to the occurrence, per single amino acid position, of sequences predicted to express authentic NadA fragments, relative to those predicted to express irrelevant or no polypeptides. The inset in figure A reports the same data with a higher y-axis magnification. The horizontal axis reports the amino acid positions of the translated NadA sequence. A, unselected library; B and C, library outputs after one and two rounds of selection, respectively. D, Cumulative enrichment factors for each amino acid position derived from NadA fragments obtained after one (blue line) and two (red line) rounds of selection; colored bars in the horizontal axis refer to NadA domains; the area between the dashed vertical lines correspond to the cell binding region of NadA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114159#pone.0114159-Tavano1" target="_blank">[18]</a>. E and F, enrichment factors of NadA fragments after one and two rounds of selection, respectively. Only the fragments laying in the upper quartile of enrichment factors values are shown.</p

Properties of the antigen-specific phage library before and after selection with a pool of serum samples from volunteers immunized with the Bexero anti-MenB vaccine.

<p>A–C, abundance of “natural frame” <i>nadA</i> fragments in the library before (A) and after the first and second rounds of selection (B and C, respectively). Each point represents the number of unique fragments (vertical axis) displaying the number of copies indicated in the horizontal axis; D–F, <i>nadA</i> fragment length distribution before (D) and after the first and second rounds of selection (E and F, respectively).</p

Schematic outline of the epitope mapping approach.

<p>The gene encoding the antigen is fragmented by DNAse digestion and the gene fragments are inserted into lambda phage vectors. The phage library is mixed with immune serum and phage particles binding to immunoglobulins are separated using Protein-G coated magnetic beads. The inserts of the phage population obtained after selection are massively sequenced and compared with those of the original unselected library using an <i>ad hoc</i> developed software which identifies the region(s) of the antigen targeted by serum antibodies.</p

Immunoreactivity of selected phage clones corresponding to different fragments of the <i>N. meningitidis</i> NadA antigen.

<p>Immunoreactivity of selected phage clones corresponding to different fragments of the <i>N. meningitidis</i> NadA antigen.</p

Inhibition of the candidacidal activity of sera from animals immunized with plasmids containing selected peptide sequences (pT.TK1, left panel and pT.TK4, right panel) by laminarin, a soluble β-1,3-glucan, pustulan, a soluble β-1,6-D-glucan, and keyhole limpet hemocyanin (KLH)-conjugated peptides.

<p>Inhibition of the candidacidal activity of sera from animals immunized with plasmids containing selected peptide sequences (pT.TK1, left panel and pT.TK4, right panel) by laminarin, a soluble β-1,3-glucan, pustulan, a soluble β-1,6-D-glucan, and keyhole limpet hemocyanin (KLH)-conjugated peptides.</p

Binding of mAb K20 to <i>Candida albicans</i> cell wall, particularly on germ tubes and budding cells.

<p>MAb K20 labeled with biotin was used in the immunofluorescence assay with streptavidin-fluorescein.</p

Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein-0

<p><b>Copyright information:</b></p><p>Taken from "Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein"</p><p>BMC Cancer 2006;6():41-41.</p><p>Published online 24 Feb 2006</p><p>PMCID:PMC1402309.</p><p>Copyright © 2006 Pavoni et al; licensee BioMed Central Ltd.</p>uration library was constructed by error-prone PCR method

Plasmids used for immunization studies.

<p>*pT.neo is a previously described plasmid espression vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105727#pone.0105727-Beninati2" target="_blank">[26]</a> used for insertion of the selected peptide sequences (plasmids pT.TK1–4),</p><p>**pmIFN-γ is a previously described plasmid encoding for murine interferon-γ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105727#pone.0105727-LoPasso1" target="_blank">[27]</a> used for co-immunization studies.</p><p>Letters in bold indicate the antigenic peptides selected from phage displayed libraries</p><p>Plasmids used for immunization studies.</p

Binding of phage clones to mAb K20.

<p>Plates were sensitized with a mAb directed against phage protein III (1 µg/ml) and 100 µl of purified phage clones (10¹¹ pfu/ml) were added. After 1 h incubation, mAb K20 or an isotype-matched IgM (1 µg/ml) were added. Binding was detected by using alkaline phosphatase-conjugated anti-mouse IgM Ab. Pools of libraries, (pVIII-12aa/pVIII.Cys-Cys or pVIII-9aa.Cys/pVIII.Cys-Cys or pVIII-9aa/pVIII-9aa.Cys) were used in different phage selections. Results are reported in separate panels (A, B or C). Phage pC89, displaying wild-Type pVIII, was used as negative control. Data represents the means ± the SD of three determinations.</p