Production and Characterization of Polyclonal Anti-ldiotypic Anti-TRH Antibodies: Application to the Study of Pituitary TRH Receptor
| Autor: | Pierre-André Cazenave, Andrée Tixier-Vidal, D. Gourdji, Philippe Denoulet, Dominique Grouselle, Annie Rousselet, Jacques Roland |
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| Rok vydání: | 1994 |
| Předmět: |
endocrine system
medicine.medical_specialty endocrine system diseases Endocrinology Diabetes and Metabolism Immunoblotting Fluorescent Antibody Technique Thyrotropin-releasing hormone Cellular and Molecular Neuroscience Endocrinology Antibody Specificity Thyrotropic cell Internal medicine Tumor Cells Cultured medicine Animals Pituitary Neoplasms Receptor Thyrotropin-Releasing Hormone biology Endocrine and Autonomic Systems Receptors Thyrotropin-Releasing Hormone Radioimmunoassay Saponins Molecular biology Prolactin Antibodies Anti-Idiotypic Rats Polyclonal antibodies Growth Hormone Pituitary Gland biology.protein Antibody hormones hormone substitutes and hormone antagonists Immunostaining |
| Zdroj: | Neuroendocrinology. 59:495-504 |
| ISSN: | 1423-0194 0028-3835 |
| DOI: | 10.1159/000126696 |
| Popis: | cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteinsor = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells. |
| Databáze: | OpenAIRE |
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