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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.


Related in: MedlinePlus

Immunocytochemistry on four cell types. The cell lines are: (A) Immortalised human dermal microvascular endothelial cells (HMEC-1), (B) adult skin fibroblasts (ASF-2), (C) breast adenocarcinoma cells (MCF-7) and (D) immortalized bone marrow-derived mesenchymal cells (hMSC-TERT). The soluble scFv antibody fragment purified from clone 2E shows strong preference for endothelial cells. Cells were co-stained with DAPI and the merged pictures here shown were taken using a 63× objective lens.
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fig05: Immunocytochemistry on four cell types. The cell lines are: (A) Immortalised human dermal microvascular endothelial cells (HMEC-1), (B) adult skin fibroblasts (ASF-2), (C) breast adenocarcinoma cells (MCF-7) and (D) immortalized bone marrow-derived mesenchymal cells (hMSC-TERT). The soluble scFv antibody fragment purified from clone 2E shows strong preference for endothelial cells. Cells were co-stained with DAPI and the merged pictures here shown were taken using a 63× objective lens.

Mentions: Soluble scFv antibody fragments of the three clones 1D, 2E and 3B, were expressed and purified. ICC was performed on four different cell lines: Immortalized HMEC-1, ASF-2, breast adenocarcinoma cells (MCF-7) and immortalized bone marrow-derived mesenchymal cells (hMSC-TERT) 33,36–38. In all the performed ICC experiments, including those performed with the negative control, the MCF-7 cancer cell line displayed a weak consistent staining. This staining is likely attributed to upregulation of the proto-oncogene c-Myc in this particular cell line, and the following binding of the mouse Cy3 conjugated anti-c-Myc antibody used for detection 40,41. 2E displayed strong positive binding to the HMEC-1 cell line. Only minor reactions were found in the other three cell lines (Fig.5). This exclusive staining pattern towards endothelial cells indicates that this particular antibody fragment could be specific towards the same type of cell that it was selected against. 3B did not provide any positive staining in any of the four cell types, despite repeated attempts. 1D showed a weak binding to the HMEC-1 cell line and none in the other three cell lines (data not shown). For both antibodies the presence of full length tag sequences was verified by Western blotting as described in 42 (data not shown). The specificity of the three antibody fragments was further tested by IHC.


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)

Immunocytochemistry on four cell types. The cell lines are: (A) Immortalised human dermal microvascular endothelial cells (HMEC-1), (B) adult skin fibroblasts (ASF-2), (C) breast adenocarcinoma cells (MCF-7) and (D) immortalized bone marrow-derived mesenchymal cells (hMSC-TERT). The soluble scFv antibody fragment purified from clone 2E shows strong preference for endothelial cells. Cells were co-stained with DAPI and the merged pictures here shown were taken using a 63× objective lens.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Immunocytochemistry on four cell types. The cell lines are: (A) Immortalised human dermal microvascular endothelial cells (HMEC-1), (B) adult skin fibroblasts (ASF-2), (C) breast adenocarcinoma cells (MCF-7) and (D) immortalized bone marrow-derived mesenchymal cells (hMSC-TERT). The soluble scFv antibody fragment purified from clone 2E shows strong preference for endothelial cells. Cells were co-stained with DAPI and the merged pictures here shown were taken using a 63× objective lens.
Mentions: Soluble scFv antibody fragments of the three clones 1D, 2E and 3B, were expressed and purified. ICC was performed on four different cell lines: Immortalized HMEC-1, ASF-2, breast adenocarcinoma cells (MCF-7) and immortalized bone marrow-derived mesenchymal cells (hMSC-TERT) 33,36–38. In all the performed ICC experiments, including those performed with the negative control, the MCF-7 cancer cell line displayed a weak consistent staining. This staining is likely attributed to upregulation of the proto-oncogene c-Myc in this particular cell line, and the following binding of the mouse Cy3 conjugated anti-c-Myc antibody used for detection 40,41. 2E displayed strong positive binding to the HMEC-1 cell line. Only minor reactions were found in the other three cell lines (Fig.5). This exclusive staining pattern towards endothelial cells indicates that this particular antibody fragment could be specific towards the same type of cell that it was selected against. 3B did not provide any positive staining in any of the four cell types, despite repeated attempts. 1D showed a weak binding to the HMEC-1 cell line and none in the other three cell lines (data not shown). For both antibodies the presence of full length tag sequences was verified by Western blotting as described in 42 (data not shown). The specificity of the three antibody fragments was further tested by IHC.

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.


Related in: MedlinePlus