Sandwich Immunoassay for Bioactive Plasma Adrenomedullin

Adrenomedullin (ADM) is a circulating peptide known to regulate vasodilation and vascular integrity. Increased plasma ADM concentrations have been described for several life-threatening conditions, including cardiovascular diseases and septic shock. Reliable methods for the simple quantification of bioactive ADM (bio-ADM) are lacking.

Monoclonal antibodies against the amidated C-terminus and middle portion of bio-ADM were generated and used for the development of a 1-step immunometric assay for the specific quantification of bio-ADM in plasma. The assay was developed in a microtiter plate/chemiluminescence label format with a significantly reduced incubation time. Precision, linearity, specimen stability, and distribution of results in healthy subjects were evaluated.

The use of monoclonal antibodies against predetermined epitopes of bio-ADM enabled the development of an assay for the determination of bio-ADM directly in EDTA plasma. Plasma samples were stable for up to 24 h at ambient temperature and over multiple freeze-thaw cycles without loss of immunoreactivity. The assay had a limit of detection of 3 pg/mL and a limit of quantification of 11 pg/mL. The assay exhibited acceptable linearity characteristics and was not influenced by complement factor H, a putative ADM-binding protein. In healthy subjects, bio-ADM concentrations were all above the limit of detection, and approximately half of them were above the limit of quantification.

By using monoclonal antibodies with defined epitope specificities, we have developed a simple, rapid, accurate, and sensitive sandwich immunoassay for bio-ADM. The assay is a potentially novel tool to support patient management, particularly in acute care in the field of sepsis and other indications, which are currently being investigated, such as acute heart failure.