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

Schematic presentation of the structure of TRIM28.

<p>The conserved RBCC motif is present at the N-terminal domain; the variable C-domain includes the PHD and BROMO domains. Image adapted from Hatakeyama <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113688#pone.0113688-Hatakeyama1" target="_blank">[45]</a>. Author's approval was obtained for the use of this image; original image is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113688#pone.0113688.s002" target="_blank">Figure S2</a>.</p

Schematic presentation of the whole workflow.

<p>1. Material preparation – expanding a GBM cell line, laminin grown and enriched in stem-like cells, 2. Llama immunization with whole GBM cells, 3. Construction of a nanobody library, 4. Phage display cycle – enrichment of antigen specific nanobodies on various biological samples (whole protein extract from GBM tissues, membrane protein-enriched fractions from GBM tissues and GBM stem-like cell lines), 5. ELISA - selection of GBM specific nanobodies, 6. Nanobody production – large scale production and purification of specific nanobodies, 7. Nanobody:antigen pairs – immobilizing the nanobodies and binding to the corresponding antigens, 8. Antigen identification by mass spectrometry, 9. Antigen validation by Western blot. See Materials and methods for additional experimental details.</p

Antigen validation.

<p>Western blot membrane after probing with a monoclonal anti-GAPDH antibody (36 kDa band) used as an internal control for amount of protein loaded in each lane, a monoclonal anti-β-actin antibody (48 kDa band) and monoclonal anti-TRIM28 antibody (100 kDa band). Samples: GSCm – membrane protein-enriched extract isolated from GBM stem-like cell lines; GBMm – membrane protein-enriched extract isolated from GBM tissues; NBTm – membrane protein-enriched extract isolated from human brain samples; GSCc – cytosolic/nuclear protein fraction isolated from GBM stem-like cell lines; GBMc – cytosolic/nuclear protein fraction isolated from GBM tissues; NBTc – cytosolic/nuclear protein fraction isolated from human brain samples; GBMw – whole protein extract from GBM tissues; NBTw – whole protein extract from human brain samples.</p

Relative band intensities of antigens validated with Western blot.

<p>The relative band intentisy of the antigens, TRIM28 and β-actin, was calculated as the ratio between arbitrary units of the band of the antigen and the arbitrary units of the band of the internal control, GAPDH [AU(antigen)/AU(GAPDH)]. Samples: GSCc – cytosolic/nuclear protein fraction isolated from GBM stem-like cell lines; GBMc – cytosolic/nuclear protein fraction isolated from GBM tissues; NBTc – cytosolic/nuclear protein fraction isolated from human brain samples.</p

Sequences of the nanobodies that were chosen for the large-scale production.

<p>The amino acid sequence of the CDR3 regions of the nanobodies is given in alphabetic order.</p