A monoclonal antibody to alpha-human atrial natriuretic polypeptide.

Abstract

SUMMARY A monoclonal antibody to o-human atrial natriuretic polypeptide (a-hANP), KY-ANP-I, hasbeen produced by fusion of a nonproducmg mouse myeloma cell tine, X63-Ag8.653, with spleen ceDs from BALB/c mice immunized with synthetic a-hANP conjugated to bovine thyrogtobuhn using the carbodiimide coupling procedure. Hybrklomas were screened for antibody production by rarJiolmmunoassay using culture media and l2S I-a-hANP. They were cloned by the llmtHng dilution technique, expanded hi culture, and injected mtraperHoneaDy into BALB/c mice. The obtained antibody belonged to the immunogiobunn G, subclass. Analysis by a Scatchard plot revealed a high affinity for a-hANP, with an association constant of 3.1 x 10" M~'. With this monoclonal antibody, a specific radioimmunoassay for a-hANP has been established. The antibody in mouse ascites was available for radioimmunoassay at a final dilution of 1:10*. Values of IC| 0 and IC<, in this radioimmunoassay were 3 and 30 fmol/tube, respectively. The radioimmunoassay showed a cross-reactivity of 0.9% with a-rat ANP. a-hANP-<8-22) and a-ANP-(l-6) exhibited less cross-reactivity than a-rat ANP on a molar basis. There was no cross-reaction with a-ANP-(17-28). Thus, the recognized epitope must be located in the N-terminal half of the ring structure of a-hANP including Met 12 residue. This radioimmunoassay could detect y-hANP and 0-hANP as well as a-hANP. The monoclonal antibody was also useful for unmunohistochemical studies. ANP-positive cells were finely stained hi the human atrium using the avidin-btotin-peroxidase complex technique. These results indicate that this monoclonal antibody to a-hANP will become a powerful tool for investigating the physiological and pathophvsiok>gkal significance of ANP. uretic polypeptides (ANPs), also known as atrial natriuretic factor (ANF), was isolated from human and rat atrial tissues and implicated in the control of water and electrolyte balance and blood pressure. kidney," and lung. 12 This antiserum against a-ANP was also used for immunohistochemical studies.

Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide.

“…We previously reported monoclonal antibodies to ANP (32,53) and their useful applications, including immunohistochemistry (32, 54-56) and a highly sensitive sandwich enzyme immunoassay for plasma a-hANP (57,58). In addition, they were used for a neutralization experiment to block endogenous ANP in hypertensive rats, resulting in the aggravation ofhypertension (59).…”

Gene and Phenotype Analysis of Congenital Generalized Lipodystrophy in Japanese: A Novel Homozygous Nonsense Mutation in Seipin Gene

“…The presence of hypertrophic cardiomyopathy was assessed using echocardiography, electrocardiography, and plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels. Plasma ANP and BNP levels were determined by immunoradiometric assay (Shionogi, Osaka, Japan) (16,17). Fatty liver was diagnosed by both ultrasound and computed tomographic imaging.…”

Ventricular expression of atrial natriuretic polypeptide and its relations with hemodynamics and histology in dilated human hearts. Immunohistochemical study of the endomyocardial biopsy specimens.

“…"28 The A monoclonal antibody to a-human ANP was prepared as previously described. 35 The recognized epitope of the monoclonal antibody is located in the N-terminal half of the ring structure of a-human ANP including Met'2 residue. This radioimmunoassay can recognize not only a-human ANP but also ,3-human ANP with a cross-reactivity of 80% on a molar basis and y-human ANP on an equimolar basis.…”

“…This radioimmunoassay can recognize not only a-human ANP but also ,3-human ANP with a cross-reactivity of 80% on a molar basis and y-human ANP on an equimolar basis. 35 Immunohistochemical reactions according to an indirect immunoperoxidase method36 were performed with some technical modifications. In the first step, intrinsic peroxidase activity was inhibited by the addition of 0.3% hydrogen peroxidase in 0.01 mol/1 phosphate-buffered saline (PBS), and nonspecific binding was blocked with normal goat serum.…”