Sandwich immunoassay for adrenomedullin precursor and its practical application

Abstract

Adrenomedullin (ADM) is a multifaceted peptide hormone involved in numerous physiological processes, including vascular stability, vasodilation, angiogenesis, and anti-inflammatory responses. The processing of ADM results in several fragments, including midregional proadrenomedullin (MR-proADM), and glycine-extended ADM (ADM-Gly) and bioactive ADM (bio-ADM). MR-proADM, the stable ADM fragment, and bio-ADM, the active form of ADM with a short half-life, have been shown to be potent biomarkers in a variety of pathologies. ADM-Gly, the direct precursor of bio-ADM, is a predominant form in human plasma, but remains less understood and least investigated. This study presents the development of a specific immunoluminometric assay for the quantification of ADM-Gly and offers a robust one-step approach for large-scale sample screening. Applied to human and rodent plasma, it elucidates the release kinetics and plasma half-life of ADM-Gly. Our findings confirm the predominance of ADM-Gly in healthy individuals and its significant release under pathological conditions. Our immunoluminometric assay enables precise measurement of ADM-Gly, advancing research into ADM-related pathophysiology and supporting its use as a biomarker and therapeutic target in various diseases.

Keywords: ADM-Gly; Adrenomedullin; C-terminal amidation; C-terminally glycine extended peptide hormones.

Fig. 1

Schematic representation of Adrenomedullin processing (top) and quantification of ADM-Gly (bottom). Top: Proteolytic processing of pro-Adrenomedullin results in generation of PAMP-Gly (pro-Adrenomedullin N-terminal 20 Peptide), MR-proADM, ADM-Gly and CT-proADM (C-terminal pro-Adrenomedullin fragment). ADM-Gly and PAMP-Gly are partially amidated. Components highlighted by the dashed box are secreted into the circulation. Bottom: Schematic representation of the ADM-Gly assay setup. Both, ADM-Gly and bio-ADM are present in the analyzed sample and both peptides interact during incubation (20 h at 2–8 °C and 600 rpm) with the capture antibody (green). ADM-Gly is recognized by the MACN labelled tracer antibody (blue) that does not cross-react with bio-ADM. After washing of unbound components, a MACN derived luminescence signal is generated.

Fig. 2

(A) Time dependent change of ADM-Gly und bio-ADM in human plasma samples after activation of endogenous PAM. Elevation of bio-ADM (open bars) and depletion of ADM-Gly (grey bars) is shown as % of total ADM. Total ADM is shown in pg/mL (open circles, bold line). Each bar represents a mean of 4 independent heparin samples. (B) Recovery of ADM-Gly from a mixture of bio-ADM and ADM-Gly. (C) Representative dose response curve of the ADM-Gly calibrator (closed circles, dashed line) and interassay precision profile (open circles, bold line). Each point of the interassay precision profile represents a mean of 16 measurements. (D) Ex vivo half-life fit of synthetic human ADM-Gly in fresh human serum, determined from on-phase decay model.

Fig. 3

(A–C) Analysis of ADM-Gly, bio-ADM and ADM ratio in n = 128 EDTA plasma samples from self-reported healthy individuals. Frequency distributions of ADM-Gly (A), bio-ADM (B), and ADM-Gly/bio-ADM ratio (C) with normal distribution curves (solid red lines). (D–F) Analysis of bio-ADM (D), ADM-Gly (E) and ADM ratio (F) in a CLP model in mice. Sham animals are shown in grey, CLP animals are shown in red. At baseline (BL) data of sham and CLP animals is combined in one bar. At days 2–7, data from n = 6 sham animals and n = 5 CLP animals is shown in each bar. Significancy was tested with Kruskal-Wallis test (Dunn’s correction for multiple comparisons). *p = 0.011–0.036; ***p = 0.0008.