Enzyme immunoassay for alpha-human atrial natriuretic polypeptide--direct measurement of plasma level.

A sandwich enzyme immunoassay for alpha-human atrial natriuretic polypeptide (alpha-hANP) was developed. Polystyrene balls were coated with monoclonal IgG1 specific for the N-terminal half of the ring structure of alpha-hANP, and rabbit Fab' specific for the C-terminal (17-28) of alpha-hANP was conjugated to horseradish peroxidase. The polystyrene ball was incubated with alpha-hANP standards or plasma and subsequently with the conjugate. Bound peroxidase activity was measured by fluorimetry. No addition of hANP-free plasma for the standard curve was required when less than 50 microliters of plasma sample was used. The detection limit of alpha-hANP was 30 fg (10 amol). Using 50 microliters of plasma, we could detect 0.6 ng (0.2 pmol)/l without extraction. The basal plasma alpha-hANP level of healthy men in a supine position after an overnight fast was 24.5 +/- 13.2 (SD) ng/l and tended to decrease (15.3 +/- 8.5 (SD) ng/l) after intravenous administration of furosemide (40 mg) and subsequent one hour walking. There was a highly significant correlation between the plasma alpha-hANP concentrations measured by this enzyme immunoassay and by radioimmunoassay (r = 0.92). This enzyme immunoassay for alpha-hANP allows measurement of plasma alpha-hANP without extraction even in the volume-contracted state.