A monoclonal antibody to α-human atrial natriuretic polypeptide

M. Mukoyama, K. Nakao, H. Sugawa, N. Morii, A. Sugawara, T. Yamada, H. Itoh, S. Shiono, Y. Saito, H. Arai, T. Mori, H. Yamada, Y. Sano, H. Imura

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Abstract

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.

Original languageEnglish
Pages (from-to)117-121
Number of pages5
JournalHypertension
Volume12
Issue number2
DOIs
Publication statusPublished - 1988 Jan 1
Externally publishedYes

ASJC Scopus subject areas

  • Internal Medicine

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    Mukoyama, M., Nakao, K., Sugawa, H., Morii, N., Sugawara, A., Yamada, T., Itoh, H., Shiono, S., Saito, Y., Arai, H., Mori, T., Yamada, H., Sano, Y., & Imura, H. (1988). A monoclonal antibody to α-human atrial natriuretic polypeptide. Hypertension, 12(2), 117-121. https://doi.org/10.1161/01.HYP.12.2.117