Production of polyclonal antibody specific for human natriuretic peptide receptor B
| Autor: | Kayoko Kawashima, Takaaki Nasu, Reiko Izumi, Kazuhiro Nagahira, Yoshiaki Fukuda, Toshihiro Nakanishi, Chika Noguchi, Katsuhiko Kitano |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Immunogen
Recombinant Fusion Proteins Blotting Western Immunology CHO Cells Transfection Antibodies Antigen Antibody Specificity Cricetinae Animals Humans Immunology and Allergy Cyclic GMP Direct fluorescent antibody Antiserum Vaccines Synthetic biology Fusion protein Molecular biology Immunoglobulin Fc Fragments Solubility Biochemistry Guanylate Cyclase Polyclonal antibodies Antibody Formation biology.protein Rabbits Antibody Immunoglobulin Heavy Chains Protein A Receptors Atrial Natriuretic Factor |
| Zdroj: | Journal of Immunological Methods. 194:147-153 |
| ISSN: | 0022-1759 |
| DOI: | 10.1016/0022-1759(96)00075-0 |
| Popis: | Polyclonal antibody against human natriuretic peptide receptor B (NPR-B) was produced using as immunogen a soluble chimeric protein consisting of the extracellular domain of the receptor fused with Fc portion of human IgG. The antibody was purified with protein A column, and then subjected to an adsorption of anti-Fc antibody using IgG column. The purified antibody recognized human NPR-B but not the related receptor NPR-A. The antibody inhibited C-type natriuretic peptide (CNP)-mediated intracellular cGMP accumulation in a dose-dependent manner. With regard to specific activity for the neutralization, the antibody purified with IgG column was significantly stronger than that before the adsorption step, indicating that the purification of the antibody with IgG column was extremely effective to remove the contaminating anti-Fc antibody from anti-NPR-B antibody. Western blot analysis using the purified antibody revealed that while the native NPR-B exists as an oligomer, the truncated NPR-B lacking most of its cytoplasmic domain is a monomer. This finding suggests that the cytoplasmic region may be involved in the oligomerization of the receptor. The results in this study demonstrate that soluble IgG fusion protein is very effective and useful for generating specific antibodies to the proteins expressed on cell surface. |
| Databáze: | OpenAIRE |
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