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 consisti ng of the extracellular domain of the receptor fused with Fc portion o f 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)-mediate d intracellular cGMP accumulation in a dose-dependent manner. With reg ard to specific activity for the neutralization, the antibody purified with IgG column was significantly stronger than that before the adsor ption step, indicating that the purification of the antibody with IgG column was extremely effective to remove the contaminating anti-Fc ant ibody from anti-NPR-B antibody. Western blot analysis using the purifi ed antibody revealed that while the native NPR-B exists as an oligomer , the truncated NPR-B lacking most of its cytoplasmic domain is a mono mer. This finding suggests that the cytoplasmic region may be involved in the oligomerization of the receptor. The results in this study dem onstrate that soluble IgG fusion protein is very effective and useful for generating specific antibodies to the proteins expressed on cell s urface.