A monoclonal antibody to alpha-human atrial natriuretic polypeptide

A monoclonal antibody to alpha-human atrial natriuretic polypeptide (alpha-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 alpha-hANP conjugated to bovine thyroglobulin using the carbodiimide coupling procedure. Hybridomas were screened for antibody production by radioimmunoassay using culture media and 125I-alpha-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 alpha-hANP, with an association constant of 3.1 x 10(10) M-1. With this monoclonal antibody, a specific radioimmunoassay for alpha-hANP has been established. The antibody in mouse ascites was available for radioimmunoassay at a final dilution of 1:10(6). 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 alpha-rat ANP. alpha-hANP-(8-22) and alpha-ANP-(1-6) exhibited less cross-reactivity than alpha-rat ANP on a molar basis. There was no cross-reaction with alpha-ANP-(17-28). Thus, the recognized epitope must be located in the N-terminal half of the ring structure of alpha-hANP including Met12 residue. This radioimmunoassay could detect gamma-hANP and beta-hANP as well as alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)