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Selection of cell-type specific antibodies on tissue-sections using phage display.

Larsen SA, Meldgaard T, Lykkemark S, Mandrup OA, Kristensen P - J. Cell. Mol. Med. (2015)

Bottom Line: To eliminate phage antibodies binding to antigens ubiquitously expressed and retrieve phage antibodies binding specifically to antigens expressed by the sub-population of cells, the area of interest is protected by a 'shadow stick'.The phage antibodies on the remaining areas on the slide are exposed to UV light, which introduces cross-links in the phage genome, thus rendering them non-replicable.In this work we applied the technology, guided by CD31 expressing endothelial cells, to isolate recombinant antibodies specifically binding biomarkers expressed either by the cell or in the microenvironment surrounding the endothelial cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.

No MeSH data available.


Phage ELISA for screening of the 40 selected phage antibodies. The ELISA was performed in duplicate on HMEC-1 cells of which the average absorbance (OD450-OD655) is shown. The results are shown in five rows with eight phage antibodies each. Furthest to the right are shown three controls performed in triplicate (row 6–8). The controls are: a confirmed unpublished endothelial cell-specific phage antibody named 8H (row 6), the KM13 helper phage (row 7) (both 1011 phages/well), and the secondary antibody alone (HRP-anti-M13) (row 8). The phage antibodies 1D (green), 2E (red), 2G (magenta) and 3B (dark purple) performed well.
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fig03: Phage ELISA for screening of the 40 selected phage antibodies. The ELISA was performed in duplicate on HMEC-1 cells of which the average absorbance (OD450-OD655) is shown. The results are shown in five rows with eight phage antibodies each. Furthest to the right are shown three controls performed in triplicate (row 6–8). The controls are: a confirmed unpublished endothelial cell-specific phage antibody named 8H (row 6), the KM13 helper phage (row 7) (both 1011 phages/well), and the secondary antibody alone (HRP-anti-M13) (row 8). The phage antibodies 1D (green), 2E (red), 2G (magenta) and 3B (dark purple) performed well.

Mentions: Two selections were performed with the Tomlinson library on FFPE breast tissue sections using the shadow stick as described. The target areas contained CD31 or vWF positive cells in blood vessels detected by fluorescence microscopy. The two selections yielded 27 and 13 clones, respectively. These 40 clones were initially screened in duplicate by phage ELISA on the endothelial cell line HMEC-1 with phage antibodies produced in 96-well format (Fig.3). As a positive control, the phage antibody 8H, which is specific for endothelial cells (not published), was included. As negative controls, the KM13 helper phage was included as well as the secondary antibody used alone (HRP-anti-M13). This ELISA screening yielded four phage antibodies: 1D, 2E, 2G and 3B, which showed considerable higher absorbance compared with negative controls. They all derived from the selection on CD31 positive cells. These phage antibodies were further analysed by a titration assay.


Selection of cell-type specific antibodies on tissue-sections using phage display.

Larsen SA, Meldgaard T, Lykkemark S, Mandrup OA, Kristensen P - J. Cell. Mol. Med. (2015)

Phage ELISA for screening of the 40 selected phage antibodies. The ELISA was performed in duplicate on HMEC-1 cells of which the average absorbance (OD450-OD655) is shown. The results are shown in five rows with eight phage antibodies each. Furthest to the right are shown three controls performed in triplicate (row 6–8). The controls are: a confirmed unpublished endothelial cell-specific phage antibody named 8H (row 6), the KM13 helper phage (row 7) (both 1011 phages/well), and the secondary antibody alone (HRP-anti-M13) (row 8). The phage antibodies 1D (green), 2E (red), 2G (magenta) and 3B (dark purple) performed well.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549044&req=5

fig03: Phage ELISA for screening of the 40 selected phage antibodies. The ELISA was performed in duplicate on HMEC-1 cells of which the average absorbance (OD450-OD655) is shown. The results are shown in five rows with eight phage antibodies each. Furthest to the right are shown three controls performed in triplicate (row 6–8). The controls are: a confirmed unpublished endothelial cell-specific phage antibody named 8H (row 6), the KM13 helper phage (row 7) (both 1011 phages/well), and the secondary antibody alone (HRP-anti-M13) (row 8). The phage antibodies 1D (green), 2E (red), 2G (magenta) and 3B (dark purple) performed well.
Mentions: Two selections were performed with the Tomlinson library on FFPE breast tissue sections using the shadow stick as described. The target areas contained CD31 or vWF positive cells in blood vessels detected by fluorescence microscopy. The two selections yielded 27 and 13 clones, respectively. These 40 clones were initially screened in duplicate by phage ELISA on the endothelial cell line HMEC-1 with phage antibodies produced in 96-well format (Fig.3). As a positive control, the phage antibody 8H, which is specific for endothelial cells (not published), was included. As negative controls, the KM13 helper phage was included as well as the secondary antibody used alone (HRP-anti-M13). This ELISA screening yielded four phage antibodies: 1D, 2E, 2G and 3B, which showed considerable higher absorbance compared with negative controls. They all derived from the selection on CD31 positive cells. These phage antibodies were further analysed by a titration assay.

Bottom Line: To eliminate phage antibodies binding to antigens ubiquitously expressed and retrieve phage antibodies binding specifically to antigens expressed by the sub-population of cells, the area of interest is protected by a 'shadow stick'.The phage antibodies on the remaining areas on the slide are exposed to UV light, which introduces cross-links in the phage genome, thus rendering them non-replicable.In this work we applied the technology, guided by CD31 expressing endothelial cells, to isolate recombinant antibodies specifically binding biomarkers expressed either by the cell or in the microenvironment surrounding the endothelial cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.

No MeSH data available.