Novel Chemiluminescent Enzyme Immunoassays for Individual Quantification of 3 Endogenous Molecular Forms of Atrial Natriuretic Peptide in Human Plasma

Abstract

Abstract Background Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are cardiac peptide hormones with pivotal roles in maintaining cardiovascular homeostasis. BNP and its precursor fragment are accepted as gold standard markers for heart failure (HF). Human ANP is present in the atria of the heart and plasma as 3 endogenous molecular forms designated α-ANP, β-ANP, and proANP. A previous study indicated that the ratios of these 3 ANP forms are altered in the plasma of HF patients. The purpose of our study was to establish immunoassays for quantifying the individual ANP forms to collect clinical information. Methods We developed 3 plate-based chemiluminescent enzyme immunoassays (CLEIAs) for measuring total ANP (i.e., sum of α-ANP, β-ANP, and proANP), β-ANP, and proANP levels. To minimize background signals, we added single-step PEGylation targeting the immobilized antibody in the conventional plate-based sandwich CLEIA procedure. Results CLEIAs with PEGylation showed sensitivity, specificity, reproducibility, and accuracy satisfying clinical requirements. Two of the CLEIAs enabled direct measurement in plasma samples. During treatments, acute decompensated HF patients exhibited marked decreases in plasma β-ANP levels but moderate decreases in plasma proANP level. The plasma ratios of α-ANP/total ANP and proANP/total ANP in acute decompensated HF patients were maintained, whereas the β-ANP/total ANP ratio was significantly decreased at discharge. Conclusions The combination of the 3 CLEIAs enabled accurate quantification of α-ANP, β-ANP, and proANP, even in plasma samples, and indicated the potential of β-ANP and proANP as circulating biomarkers for HF, with different characteristics from that of BNP.