Production of polyclonal antibody specific for human natriuretic peptide receptor B.
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.
Kitano
K
K
Suntory Institute for Biomedical Research, Osaka, Japan.
Fukuda
Y
Y
Nagahira
K
K
Nasu
T
T
Noguchi
C
C
Izumi
R
R
Kawashima
K
K
Nakanishi
T
T
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology*; D000917:Antibody Formation; D000918:Antibody Specificity; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D006152:Cyclic GMP / Q000096:biosynthesis; D006162:Guanylate Cyclase / Q000032:analysis / Q000276:immunology*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D011817:Rabbits; D017461:Receptors, Atrial Natriuretic Factor / Q000032:analysis / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012995:Solubility; D014162:Transfection; D014614:Vaccines, Synthetic / Q000494:pharmacology
D000906:Antibodies; D007141:Immunoglobulin Fc Fragments; D007143:Immunoglobulin Heavy Chains; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; C073616:atrial natriuretic factor receptor C; D006152:Cyclic GMP