A Two-site Delfia Immunoassay for Measurements of the N-terminal Peptide of pro-Atrial Natriuretic Peptide (nANP)

Abstract

A rapid, sensitive and reliable two-site immunoassay for measurements of the N-terminal peptide of pro-atrial natriuretic peptide (nANP) is presented. The method uses one monoclonal antibody, directed against the N-terminal part of nANP, as catcher antibody and another monoclonal antibody, directed against the C-terminal part of nANP, as detector antibody. The catcher antibody is biotinylated and is bound to streptavidin pre-coated microtiter strips. The detector antibody is labelled with Europium, which is measured in a Wallac DELFIA time-resolved fluorometer. Blood collected in plain Vacutainer^r` tubes gave same measured amounts of nANP as blood collected in heparinised tubes. Blood collected in tubes containing EDTA gave same measured amounts of nANP as the plain tubes, provided that a 2-step assay protocol was used. Based upon 100 healthy blood donors, a reference interval was calculated to <450 pmol/L. Within the reference group there was a significant increase of serum nANP with age. Based on 42 patients with different degree of impaired renal function, a significant correlation of nANP and serum creatinine was found. Assay performance, given as total assay variation was 12%, 10% and 9% respectively at serum levels of 140, 970 and 3500 pmol/L. It is concluded that this method is fast, sensitive and reliable for clinical measurements of nANP.