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).


METHODS
PAMP was reacted simultaneously with 2,4-dinitrophenyl (DNP)-biotinyl-bovine serum albumin (BSA)-anti-PAMP Fab' conjugate and anti-PAMP Fab'-beta-D-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 microl 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.
Hashida
Seiichi
S
Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan. shashida@fc.miyazaki-med.ac.jp
Kitamura
Kazuo
K
Nagatomo
Yoshitatsu
Y
Shibata
Yoshisato
Y
Imamura
Takuroh
T
Yamada
Kazuhiro
K
Fujimoto
Shouichi
S
Kato
Johji
J
Morishita
Kazuhiro
K
Eto
Tanenao
T
D053607:Adrenomedullin; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D002138:Calibration; D002318:Cardiovascular Diseases / Q000097:blood*; D003429:Cross Reactions; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000097:blood* / Q000276:immunology; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000032:analysis
D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D053607:Adrenomedullin; D001616:beta-Galactosidase