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 250pM, and the minimum detection limit was 0.13pM, 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.