Homogeneous time-resolved fluoroimmunoassay for the measurement of midregional proadrenomedullin in plasma on the fully automated system B.R.A.H.M.S KRYPTOR®
Introduction
Adrenomedullin (ADM) is one of the most potent vasodilators [1] and elevated concentrations have been reported in chronic and acute cardiac diseases, in respiratory tract infections and in sepsis [2]. The measurement of the mature hormone is technically challenging [3] due to its short half-life time of 22 min in the circulation [4]. A stable midregional fragment of pro-adrenomedullin (MR-proADM) consisting of the 45–92 amino acids sequence of the preproadrenomedullin was recently identified [5]. A manual immunoluminometric assay for the measurement of MR-proADM has been described previously [6]. With this assay, MR-proADM has been shown as a potential marker for prognosis in sepsis [7], in low respiratory tract diseases such as community-acquired pneumonia [8] and in cardiac dysfunction [9].
The B.R.A.H.M.S KRYPTOR® is a fully automated homogeneous random access platform. The system uses the Time Resolved Amplified Cryptate Emission (TRACE) technology [10]. This method is based on a non radiative energy transfer between a donor, the long lived fluorophore Europium Cryptate (EuC) and an acceptor, the Cyanine 5 (Cy5). The energy transfer depends on the proximity and the overlap of spectrum of the two fluorophores. In a sandwich immunoassay, the EuC and Cy5 are bound to specific antibodies. The EuC is excited at 337 nm by a nitrogen laser and the energy is transferred to the Cy5 which reemits specifically at 665 nm. This homogeneous assay uses time resolved measurements and spectral selection to eliminate the background signal of the media and to select the specific signal. Here we described a new assay for the measurement of MR-proADM using the TRACE technology on the fully automated system B.R.A.H.M.S KRYPTOR.
Section snippets
Assay
This homogeneous sandwich fluoroimmunoassay can be used equally on the two automates KRYPTOR and KRYPTOR compact (B.R.A.H.M.S AG, Hennigsdorf/Berlin, Germany). Purified anti-MR-proADM sheep polyclonal antibody [6] specific for amino-acids 68–86 (RPQDMKGASRSPEDSSPD) was coupled to Cy5 fluorophore (General Electric Healthcare). Purified anti-MR-proADM sheep polyclonal antibody [6] specific for amino-acids 83–94 (SSPDAARIRVKR) was coupled to europium cryptate TBP-mono-MP (CISBIO, Bagnols/Cèze,
Results
The direct measuring range of the assay is from 0 to 10 nmol/L. No high-dose hook effect was observed up to 225 nmol/L. Samples from 10 nmol/L and up to 100 nmol/L were automatically detected as out of range samples and diluted by the machine with the diluent of the kit at the appropriate dilution factor (1/10 or 1/100). The limit of detection (LoD) was determined at 0.05 nmol/L and the limit of quantitation at 0.23 nmol/L. The repeatability (within-run) CV was lower than 20% in the range
Discussion
We have developed a new and fully automated sandwich assay for the reliable quantification of mid-regional fragment of pro-adrenomedullin in EDTA plasma. This assay offers fast and reproducible measurements of the release of adrenomedullin in the circulation. This assay is based on the TRACE technology, a time-resolved fluorescence method in a homogeneous phase without any separation or washing steps leading to a rapid and easy to handle assay. The MR-proADM assay requires only 29 min of
Acknowledgments
We thank Nicolas Bourgoin, Céline Devos, Fanny Heurtault and Amélie Roux for excellent technical assistance.
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