Preparation of monoclonal antibodies against atrial natriuretic polypeptide precursor and application to highly sensitive sandwich enzyme immunoassay

We produced three monoclonal antibodies against different epitopes of the precursor form of atrial natriuretic polypeptide (ANP), γ -human ANP (γ-hANP) [human atrial natriuretic factor (ANF)-(1-126)]. Monoclonal antibodies were prepared by fusion of mouse myeloma cells X63-Ag8.653 with spleen cells from BALB/c mice immunized with synthetic α-hANP or γ -hANP-(1-25) [human ANF-(1-25)] conjugated to thyroglob- ulin. Of three monoclonal antibodies obtained, two were directed against the α -ANP sequence: one antibody (KY-ANP-I) recognizes the N-terminal half of the ring structure, while the other (KY-ANP-II) recognizes the N-terminal sequence of α -hANP. The third antibody (KY-ANP-III) was against γ -hANP-(1-25). With the aid of rabbit polyclonal anti-α-ANP-(17-28), we developed sandwich enzyme immunoassays. An enzyme immunoassay for a-hANP using KY-ANP-I and polyclonal Fab’ was very sensitive (0.01 fmol/tube) and we could detect 0.6 pg/ml of plasma α -hANP without extraction. Thus, monoclonal antibodies against γ -ANP are useful tools for investigating the significance of ANP and related peptides derived from γ-ANP.