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), gamma-human ANP (gamma-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 alpha-hANP or gamma-hANP-(1-25) [human ANF-(1-25)] conjugated to thyroglobulin. Of three monoclonal antibodies obtained, two were directed against the alpha-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 alpha-hANP. The third antibody (KY-ANP-III) was against gamma-hANP-(1-25). With the aid of rabbit polyclonal anti-alpha-ANP-(17-28), we developed sandwich enzyme immunoassays. An enzyme immunoassay for alpha-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 alpha-hANP without extraction. Thus, monoclonal antibodies against gamma-ANP are useful tools for investigating the significance of ANP and related peptides derived from gamma-ANP.