A monoclonal antibody to α-human atrial natriuretic polypeptide (α-hANP), KY-ANP-I, has been produced by fusion of a nonproducing mouse myeloma cell line, X63-Ag8.653, with spleen cells from BALB/c mice immunized with synthetic α-hANP conjugated to bovine thyroglobulin using the carbodiimide coupling procedure. Hybridomas were screened for antibody production by radioimmunoassay using culture media and 125I-α-hANP. They were cloned by the limiting dilution technique, expanded in culture, and injected intraperitoneally into BALB/c mice. The obtained antibody belonged to the immunoglobulin G1 subclass. Analysis by a Scatchard plot revealed a high affinity for α-hANP, with an association constant of 3.1 x 1010 M-1. With this monoclonal antibody, a specific radioimmunoassay for α-hANP has been established. The antibody in mouse ascites was available for radioimmunoassay at a final dilution of 1:106. Values of IC10 and IC50 in this radioimmunoassay were 3 and 30 fmol/tube, respectively. The radioimmunoassay showed a cross-reactivity of 0.9% with α-rat ANP.α-hANP-(8-22) and α-ANP-(1-6) exhibited less cross-reactivity than α-rat ANP on a molar basis. There was no cross-reaction with α-ANP-(17-28). Thus, the recognized epitope must be located in the N-terminal half of the ring structure of α-hANP including Met12 residue. This radioimmunoassay could detect γ-hANP and β-hANP as well as α-hANP. The monoclonal antibody was also useful for immunohistochemical studies. ANP-positive cells were finely stained in the human atrium using the avidin-biotin-peroxidase complex technique. These results indicate that this monoclonal antibody to α-hANP will become a powerful tool for investigating the physiological and pathophysiological significance of ANP.
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