Development of an ultrasensitive enzyme immunoassay for human proadrenomedullin N-terminal 20 peptide and direct measurement of two molecular forms of PAMP in plasma from healthy subjects and patients with cardiovascular disease

Objective: Proadrenomedullin N-terminal 20 peptide (PAMP) processed from an adrenomedullin precursor is a potent hypotensive peptide. It was anticipated that a mature form of PAMP (m-PAMP) and an intermediate PAMP-gly existed together in the blood. To measure concentrations of PAMPs in human plasma directly, we have developed a highly sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay, ICT-EIA). Design and methods: PAMP was reacted simultaneously with 2,4-dinitrophenyl (DNP)-biotinyl-bovine serum albumin (BSA)-anti-PAMP Fabʹ conjugate and anti-PAMP Fabʹ-β- -galactosidase conjugate. The immune complex that was formed was initially trapped onto a polystyrene bead coated with anti-DNP IgG, and then transferred onto a second polystyrene bead coated with streptavidin. The resulting three-component complex was then assayed fluorometrically. Results: The detection limits of ICT-EIA for both m-PAMP and PAMP-gly were 0.1 pmol/l with as little as 10 μl of plasma, and were a hundred times higher than with conventional radioimmunoassay (RIA). Using ICT-EIA, we determined that the plasma concentrations of m-PAMP and PAMP-gly in 51 healthy volunteers were 0.51 ± 0.19 and 1.15 ± 0.38 pmol/l (mean ± SD), respectively. Both plasma m-PAMP and PAMP-gly concentrations in patients with a variety of diseases, including hypertension, heart failure, chronic renal failure, and hemodialysis, were significantly higher than those in healthy subjects. In addition, both plasma m-PAMP and PAMP-gly concentrations in patients with New York Heart Association (NYHA) class I–IV heart failure were increased in proportion to clinical severity. Conclusions: These sensitive and specific ICT-EIAs may be used as a powerful tool for investigating the cardiovascular system in patients with heart failure.