Generation and Characterization of Monoclonal Antibodies against the Transcription Factor Nkx6.1

We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine β-cells in the pan...

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Veröffentlicht in:The journal of histochemistry and cytochemistry 2006-05, Vol.54 (5), p.567-574
Hauptverfasser: Pedersen, Inger L, Klinck, Rasmus, Hecksher-Sorensen, Jacob, Zahn, Stefan, Madsen, Ole D, Serup, Palle, Jorgensen, Mette C
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container_end_page 574
container_issue 5
container_start_page 567
container_title The journal of histochemistry and cytochemistry
container_volume 54
creator Pedersen, Inger L
Klinck, Rasmus
Hecksher-Sorensen, Jacob
Zahn, Stefan
Madsen, Ole D
Serup, Palle
Jorgensen, Mette C
description We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine β-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing β-cells, making this transcription factor a unique β-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/κ subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the β-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies. (J Histochem Cytochem 54:567-574, 2006)
doi_str_mv 10.1369/jhc.5A6827.2006
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Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing β-cells, making this transcription factor a unique β-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/κ subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the β-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies. 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Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing β-cells, making this transcription factor a unique β-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/κ subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the β-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies. 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subjects Animals
Antibodies, Monoclonal - biosynthesis
Antibodies, Monoclonal - isolation & purification
Blotting, Western
Embryo, Mammalian - metabolism
Enzyme-Linked Immunosorbent Assay
Homeodomain Proteins - immunology
Homeodomain Proteins - metabolism
Immunohistochemistry
Mice
Pancreas - embryology
Pancreas - metabolism
Rabbits
Transcription Factors - metabolism
title Generation and Characterization of Monoclonal Antibodies against the Transcription Factor Nkx6.1
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