Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

• Level A · Stronger Clinical Evidence
• Level B · Emerging clinical evidence with positive signals
• Level C · Early human research exploring benefits
• Level D · Scientific groundwork from lab and animal studies
• Emerging · Emerging topic under active research

Abstract (original English)

Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1-8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa

Evidence level

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

Browse all related research

Filter the research library by this study's title keywords, author, or publication year.