Удосконалення комерційної діяльності як фактор підвищення конкурентоспроможності підприємства

Additional file 5. ELISA to assess the interaction between Campylobacter -specific nanobodies and purified MOMP. The saturation binding curve of the interaction between coated MOMP (1 µg/mL) and a His-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) was obtained via ELISA. The dose-dependent inhibitory effect of a strep-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) on the interaction between His-tagged Nb84 (5.10−2 µg/mL) and MOMP (1 µg/mL), is demonstrated in the competition binding curve. Inhibition by strep-tagged (A) Nb5, (B) Nb22, (C) Nb23, (D) Nb24, (E) Nb49, (F) 84, (G) Nb15, (H) Nb32, (I) Nb34, (J) Nb45, (K) Nb48 and (L) Nb63, was assessed. The ELISA was developed with mouse anti-Histidine tag monoclonal antibody and goat anti-mouse IgG conjugated to alkaline phosphatase. The error bars represent the standard deviations

Overview of the binding of inhibitory nanobodies NbFedF6 and NbFedF7 on the surface of the F18 fimbrial adhesin FedF.

<p>Nanobodies NbFedF6 and NbFedF7 (green) are interacting with a near identical epitope at the interface of the two β-sheets that make up the immunoglobulin-like fold of FedF_15–165 (β-strands, α-helices and loops are colored respectively cyan, red and grey). The three complementary determining regions (CDRs) are colored respectively in orange, purple and yellow.</p

Inhibitory nanobodies recognize FedF_15–165 with low nanomolar affinity.

<p>Microscale thermophoresis (MST) was employed to determine the in solution affinity between Nb-FedF6, Nb-FedF7, Nb-FedF9 and Nb-FedF12 with FedF_15–165. (<b>A</b>) Typical MST measurement showing the interaction between Nb-FedF6 and FedF_15–165. Data points are indicated by black diamonds, the fit by the NT Analysis software is shown as a red line. (<b>B</b>) Overview on the determined dissociation constants (K_D) for the indicated Nb-FedF_15–165 interactions.</p

Blocking the attachment of F18 positive <i>E. coli</i> to piglet enterocytes by nanobodies.

<p>Nanobodies directed against the N-terminal domain of the FedF tipadhesin (FedF_15–165) were assayed in an <i>in vitro</i> adherence test of wild type F18-positive <i>E. coli</i> strain 107/86 to piglet intestinal enterocytes. As a negative control PBS buffer was added instead of nanobody. Bacterial cells adhering to villi were counted under a microscope and plotted as a percentage of wild type binding.</p

NbFedF9 inhibits the binding of F18 fimbriated <i>E. coli</i> to piglet enterocytes by occupying the carbohydrate binding site on the FedF surface.

<p>Left: structure of the complex between the inhibitory NbFedF9 (green) and FedF_15–165 (β-strands, α-helices and loops are colored respectively cyan, red and grey), which shows NbFedF9 interacting at the side of the FedF fold. The three complementary determining regions (CDRs) of NbFedF9 are colored respectively in orange, purple and yellow. Right: overlay of the NbFedF9-FedF_15–165 structure with the previously elucidated structure of the co-complex between FedF_15-165 and the blood group A type 1 hexasaccharide <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114691#pone.0114691-Moonens1" target="_blank">[17]</a>. Both the carbohydrate ligand and NbFedF9 compete for the same binding site on the FedF fold. Blood group A type 1 hexasaccharide is depicted in stick model with carbon, oxygen and nitrogen atoms colored respectively purple, red and blue.</p

Nanobodies that induce a conformational change in the D″-E loop do not inhibit the attachment of FedF towards the A6-1 carbohydrate.

<p>Consecutive injections of either FedF_15–165 or FedF_15–165-nanobody complexes over the sensor chip surface carrying an immobilized A6-1-human serum albumin glycoconjugate were performed. Nanobody NbFedF9, shown in the crystal structure to bind in the FedF carbohydrate binding site, completely blocks the FedF-A6-1 interaction. In contrary, nanobodies NbFedF6, NbFedF7 and NbFedF12 only slightly or not at all inhibit the binding of FedF on the A6-1 coated surface. The crystal structures show how NbFedF6 and NbFedF7 induce a conformational change in the D″-E loop but do not steric compete with A6-1 binding.</p

Blocking the attachment of F18 positive <i>E. coli</i> to piglet enterocytes by nanobodies.

<p>Nanobodies directed against the N-terminal domain of the FedF tipadhesin (FedF_15–165) were assayed in an <i>in vitro</i> adherence test of wild type F18-positive <i>E. coli</i> strain 107/86 to piglet intestinal enterocytes. As a negative control PBS buffer was added instead of nanobody. Bacterial cells adhering to villi were counted under a microscope and plotted as a percentage of wild type binding.</p