The prohormone of atrial natriuretic peptide (proANP) is a polypeptide of 126 amino acids. Mature atrial natriuretic peptide (ANP) consists of amino acids 99–126 and comprises 98% of the natriuretic peptides in the circulation (1). The N-terminal portion of proANP, termed proANP1–98 or NT-proANP, has a much longer half-life than mature ANP and has therefore been suggested to be a more reliable analyte for measurement than mature ANP (2). In addition to other established indications, proANP has recently gained much interest as a potential new marker in the field of sepsis (3), emphasizing the need for a reliable assay for this molecule.

All sandwich immunoassays developed for proANP to date use an antibody against the N-terminal region of proANP1–98 combined with a second antibody against either the midregion (4) or C-terminal region (5)(6). However, under certain conditions, the N-terminal region might be minimally accessible for antibody binding (7)(8). Despite the described long half-life of proANP, results from various competitive immunoassays as well as HPLC analyses indicate that proANP1–98 can be subject to further fragmentation (9)(10).

We developed a new sandwich immunoassay for midregional proANP (amino acids 53–90; Fig. 1A⇓ ). With this assay, which was named B.R.A.H.M.S SERISTRA® (B.R.A.H.M.S AG), we measured proANP values in 325 healthy controls.

EDTA-plasma samples were collected at a blood bank (Red Cross) from 325 consecutive healthy blood donors (age range, 18–67 years; 52.9% male) without clinical evidence of acute disease or a history of chronic illness. Blood donors with risk factors for heart failure were not enrolled in the study. Written consent was obtained from …

Immunoluminometric Assay for the Midregion of Pro-Atrial Natriuretic Peptide in Human Plasma

The long-predicted endocrine function of the heart has been proven by the discovery of atrial natriuretic peptide (atrial natriuretic factor, A-type natriuretic peptide; ANP) 20 years ago. This subsequently led to the description of a whole family of structurally similar but genetically distinct peptides, the natriuretic peptide family, which contributes to cardiovascular homeostasis. These looped peptides promote natriuresis and diuresis, act as vasodilators, and exert antimitogenic effects on cardiovascular tissues. Two members, ANP and brain natriuretic peptide (B-type natriuretic peptide; BNP) are secreted by the heart mainly in response to myocardial stretch induced by volume load. The natriuretic peptides are synthesized as preprohormones. The C-terminal endocrinological active peptides (ANP, BNP) and their N-terminal prohormone fragments are found in plasma. The natriuretic peptide system is activated to its highest degree in ventricular dysfunction. However, natriuretic peptides are increased in all patients with edematous disorders which lead to an increase in atrial tension or central blood volume, such as renal failure or ascitic liver cirrhosis. It could be demonstrated that in chronic heart failure patients and during the subacute phase of myocardial infarction, of all tested neurohormones, the cardiac natriuretic peptides were best markers to identify heart failure and the most powerful predictors of morbidity and mortality. Natriuretic peptides are independent markers for risk assessment. In comparative studies BNP was superior to ANP and its N-terminal prohormone fragments in myocardial infarction as well as in chronic heart failure patients. Less data on N-terminal proBNP (NT-proBNP) is available, but BNP and NT-proBNP appear to be equivalent markers. For primary care physicians natriuretic peptide measurement is useful to decide which patient with suspected heart failure warrants further investigation, particularly when assessment of left ventricular function is not readily available. Natriuretic peptides have an excellent negative predictive value, particularly in high risk patients. An increase in BNP is serious enough to warrant follow-up examinations. For the cardiologists the natriuretic peptides are helpful for guidance of therapy and monitoring disease course in heart failure patients and for risk stratification in heart failure and myocardial infarction.

The impact of cardiac natriuretic peptide determination on the diagnosis and management of heart failure.

Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction.

Background: The N-terminal fragments of A- and Btype natriuretic peptides (NT-proANP and NT-proBNP) are powerful markers of cardiac function. The current assays require refinement with regard to standardization with native calibrators and the ability to detect the actual circulating forms. Methods: The following peptides were prepared with recombinant methods: NT-proANP, NT-proBNP, proBNP1–108, and Tyr0-proBNP77–108. Fifteen peptides of 13–22 amino acids, spanning the sequences of NTproANP and NT-proBNP, were prepared by solid-phase peptide synthesis. Two immunoassays for NT-proANP and four for NT-proBNP were set up, each with a different epitope specificity. The assays were applied for the measurement of NT-proANP and NT-proBNP in healthy individuals and in patients with acute myocardial infarction. The circulating molecular forms were analyzed by gel-filtration and reversed-phase HPLC. Results: According to the HPLC analyses, circulating NT-proANP consists mainly of the full-length peptide, with some degradation at both ends. In contrast, circulating NT-proBNP is very heterogeneous. Most immunoreactive NT-proBNP is significantly smaller in size than NT-proBNP1–76, with truncation at both termini. The smallest fragments can be detected by assays directed at the central part of NT-proBNP only; assays directed at the ends gave 30 – 40% lower values. Despite the difference, the various assays correlated reasonably well with each other (r 2 0.77– 0.85). In patients with acute myocardial infarction, NT-proANP and NTproBNP concentrations were 1.8 –2.3 and 4.2– 4.5 times higher than in healthy individuals. The development of heart failure further increased the concentrations. Conclusions: Molecular heterogeneity of the circulating forms causes a serious risk of preanalytical errors in assays for NT-proBNP and, to a lesser extent, NTproANP. The development of a sandwich assay for NT-proBNP would be especially challenging. The most robust and reliable assays use antibodies directed at the central portions of NT-proANP or NT-proBNP. © 2004 American Association for Clinical Chemistry

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Molecular Heterogeneity Has a Major Impact on the Measurement of Circulating N-Terminal Fragments of A- and B-Type Natriuretic Peptides

Head-to-head comparison of the diagnostic utility of BNP and NT-proBNP in symptomatic and asymptomatic structural heart disease.

Several peptides derived from the N-terminal sequence of pro-atrial natriuretic peptide (proANP) have been tested successfully as markers of heart disease. We have developed specific and sensitive competitive enzyme immunoassays for fragments [1-30] and [31-67] of proANP. Antisera were raised in sheep against synthetic peptides predicted to be highly immunogenic. Binding specificity was determined by epitope mapping. Microtiter plates were coated with antibody specific for the Fc region of sheep IgG to capture the affinity-purified specific anti-proANP antibodies in an oriented and reproducible form. Synthetic proANP calibrators or diluted samples were incubated simultaneously with biotinylated peptide and binding was quantitated using streptavidin-peroxidase and TMB. Immunoreactive proANP could be measured in diluted plasma, serum and urine. The detection limits of the proANP[1-30] and proANP[31-67] assays were 2.5 and 10 pmol/l respectively. The linearity of samples diluted beyond the recommended assay conditions was good. Recoveries of added standard peptides ranged from 102 to 112%. Circulating concentrations of immunoreactive proANP in 115 healthy subjects ranged from 0.11 to 0.47 nmol/l proANP[1-30] and 0.18 to 0.79 nmol/l proANP[31-67]. In patients with cardiac disease, proANP levels were increased significantly. The reference interval of proANP[31-67] in urine was 0.09 to 1.7 nmol/l, several-fold higher than proANP[1-30] (<O.03 to 1.1 nmol/l). After storage for 6 months at -20 degrees C there was no detectable decrease in immunoreactivity.

Enzyme immunoassays for fragments (epitopes) of human proatrial natriuretic peptides.

Proatrial natriuretic peptides are proposed to be sensitive and specific markers for various stages of heart deficiency. Here we present a rapid and specific sandwich immunoassay for the measurement of proANP 1-98 in biological fluids like plasma, serum urine. No sample preparation prior to the assay is necessary. Polyclonal antibodies specific for distinct epitopes of proANP 1-98, predicted to be highly immunogenic, were raised in sheep and purified by immunoaffinity chromatography prior to use in the assay. Antigen binding sites of the antibodies were determined by epitope mapping with a set of peptide fragments. The capture antibody, specific for proANP 16-23, is coated directly onto microtiter plates. Recombinant proANP 1-98 is used as a standard. The biotinylated detection antibody, specific for proANP 80-88, is incubated simultaneously with 20microl of sample for 150 min. Signal is generated with a streptavidin-HRPO-conjugate and TMB as substrate. The detection limit of the assay is 50 pmol/l; intraassay CVs are below 5%, interassay CVs are lower than 10%. Dilution curves show good linearity, recovery of synthetic peptide ranged between 85% and 91%. Reference values from healthy volunteers range from 0 to 2000 pmol/l in human plasma. We conclude this assay is a easy to handle, quick and reliable method for routine measurement of proANP 1-98 and the presented manuscript describes the procedure and performance of this ELISA in detail.

Sandwich ELISA for proANP 1-98 facilitates investigation of left ventricular dysfunction.

Zusammenfassung: Brain natriuretisches Peptid (BNP) ist ein Mitglied der Familie der natiruretischen Peptide, die für die Homöostase des Salzund Flüssigkeitshaushalts mitverantwortlich sind und den Organismus gegen Flüssigkeitsüberschuß und hohen Blutdruck schützen. BNP wird bei der Frei Setzung, aus dem Ventrikel in die biologisch aktive Form BNP 32 und ein 76 Aminosäuren langes N-terminales Fragment proBNP (1-76) gespalten. Plasmakonzentrationen von BNP sind bei Patienten mit Herz-, Nierenund Lebererkrankungen sowie bei verschiedenen Formen des angeborenen und erworbenen Bluthochdrucks deutlich erhöht. In der vorliegenden Mitteilung werden der Aufbau und die Leistungscharakteristika von kompetitiven Immunoassays zur Bestimmung der proBNP Fragmente (8-29) und (32-57) beschrieben. Mit diesen Assays werden weitere Untersuchungen über zirkulierende Formen von proBNP ermöglicht.

A. Missbichler

Papers

Enzyme immunoassays for fragments (epitopes) of human proatrial natriuretic peptides.

Sandwich ELISA for proANP 1-98 facilitates investigation of left ventricular dysfunction.

Enzymimmunoassays für proBNP Fragmente (8-29) und (32-57).

Enzyme binding protein assay for 25-hydroxyvitamin d