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

被引:5
作者
Hashida, S [1 ]
Kitamura, K
Nagatomo, Y
Shibata, Y
Imamura, T
Yamada, K
Fujimoto, S
Kato, J
Morishita, K
Eto, T
机构
[1] Miyazaki Med Coll, Dept Biochem, Kiyotake, Miyazaki 8891692, Japan
[2] Miyazaki Med Coll, Dept Internal Med 1, Miyazaki 8891692, Japan
[3] Miyazaki Med Assoc Hosp, Miyazaki 8800834, Japan
关键词
enzyme immunoassay; proadrenomedullin N-terminal 20 peptide; adrenomedullin; hypertension; heart failure; human plasma; beta-D-galactosidase; Fab;
D O I
10.1016/j.clinbiochem.2003.09.007
中图分类号
R446 [实验室诊断]; R-33 [实验医学、医学实验];
学科分类号
1001 ;
摘要
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 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 mul of plasma, and were a hundred times higher than with conventional radioirnmunoassay (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. (C) 2003 The Canadian Society of Clinical Chemists. All rights reserved.
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页码:14 / 21
页数:8
相关论文
共 21 条
[1]   Glycine-extended adrenomedullin exerts vasodilator effect through amidation in the rat aorta [J].
Cao, YN ;
Kitamura, K ;
Ito, K ;
Kato, J ;
Hashida, S ;
Morishita, K ;
Eto, T .
REGULATORY PEPTIDES, 2003, 113 (1-3) :109-114
[2]  
Eto T, 1996, KIDNEY INT, pS148
[3]   A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides [J].
Eto, T .
PEPTIDES, 2001, 22 (11) :1693-1711
[4]   Differential hormonal profiles of adrenomedullin and proadrenomedullin N-terminal 20 peptide in patients with heart failure and effect of treatment on their plasma levels [J].
Etoh, T ;
Kato, J ;
Takenaga, M ;
Imamura, T ;
Kitamura, K ;
Koiwaya, Y ;
Eto, T .
CLINICAL CARDIOLOGY, 1999, 22 (02) :113-117
[5]   ANTIBODIES TO BRADYKININ + ANGIOTENSIN - USE OF CARBODIIMIDES IN IMMUNOLOGY [J].
GOODFRIEND, TL ;
LEVINE, L ;
FASMAN, GD .
SCIENCE, 1964, 144 (362) :1344-&
[6]   A HIGHLY SENSITIVE SANDWICH ENZYME-IMMUNOASSAY OF HUMAN GROWTH-HORMONE IN SERUM USING AFFINITY-PURIFIED ANTI-HUMAN GROWTH-HORMONE FAB - HORSERADISH-PEROXIDASE CONJUGATE [J].
HASHIDA, S ;
NAKAGAWA, K ;
YOSHITAKE, S ;
IMAGAWA, M ;
ISHIKAWA, E ;
ENDO, Y ;
OHTAKI, S ;
ICHIOKA, Y ;
NAKAJIMA, K .
ANALYTICAL LETTERS PART B-CLINICAL AND BIOCHEMICAL ANALYSIS, 1983, 16 (01) :31-44
[7]  
Hashida S, 1995, Biotechnol Annu Rev, V1, P403
[8]   DETECTION OF ONE MILLIATTOMOLE OF FERRITIN BY NOVEL AND ULTRASENSITIVE ENZYME-IMMUNOASSAY [J].
HASHIDA, S ;
ISHIKAWA, E .
JOURNAL OF BIOCHEMISTRY, 1990, 108 (06) :960-964
[9]   Earlier detection of human immunodeficiency virus type 1 p24 antigen and immunoglobulin G and M antibodies to p17 antigen in seroconversion serum panels by immune complex transfer enzyme immunoassays [J].
Hashida, S ;
Ishikawa, S ;
Hashinaka, K ;
Nishikata, I ;
Oka, S ;
Ishikawa, E .
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, 2000, 7 (06) :872-881
[10]   Molecular forms of circulating adrenomedullin in patients with congestive heart failure [J].
Hirayama, N ;
Kitamura, K ;
Imamura, T ;
Kato, J ;
Koiwaya, Y ;
Tsuji, T ;
Kangawa, K ;
Eto, T .
JOURNAL OF ENDOCRINOLOGY, 1999, 160 (02) :297-303