title: Direct chemiluminescent enzyme immunoassay for atrial natriuretic peptide in mammalian plasma using a PEGylated antibody.
abstract: Atrial natriuretic peptide (ANP) is a peptide hormone that is synthesized and secreted by cardiac tissues and plays a pivotal role in maintaining cardiovascular homeostasis. Clinically, ANP is used as a marker of cardiovascular diseases, including heart failure. Although multiple ANP assays are currently available, a more sensitive assay is required for the direct measurement of plasma ANP where there is limited plasma availability, especially in mouse experiments. In the current study, we developed a plate-based sandwich chemiluminescent enzyme immunoassay for the measurement of plasma ANP in rats and mice without the need for prior extraction. To minimize nonspecific binding, we performed a single-step PEGylation procedure targeting the immobilized antibody, which markedly improved the assay's sensitivity and linearity. The linear range was 0.1 to 250 pM, and the minimum detection limit was 0.13 pM, 5-fold lower than the lowest value of the commercially available kits. ANP was directly measured in plasma samples without detectable cross-reactivity with B- and C-type natriuretic peptides. The accuracy of the assay was confirmed by spike recovery tests and dilution tests and by comparison with a conventional radioimmunoassay. Based on the species cross-reactivity, this assay can be used to measure human ANP.
authors.affiliation: Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nagai
authors.affiliation: Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan. Electronic address: minamino@ncvc.go.jp.
authors.forename: Naoto
authors.initials: N
authors.lastname: Minamino
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology*; D009320:Atrial Natriuretic Factor / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D001774:Blood Chemical Analysis / Q000379:methods*; D003429:Cross Reactions; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008163:Luminescent Measurements; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D015203:Reproducibility of Results
chemical_list: D055910:Antibodies, Immobilized; D000939:Epitopes; D011092:Polyethylene Glycols; D009320:Atrial Natriuretic Factor
keywords: Atrial natriuretic peptide; Chemiluminescent enzyme immunoassay; Direct plasma measurement; PEGylation