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

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 NEP fragment which was not apparent with PE pAb, as well as a common ~ 57-60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.

Finding a reliable assay for soluble neprilysin.

BACKGROUND: Lack of validation and standardization of research-use-only (RUO) immunoassays brings with it inherent threats to authenticity and functional quality. Poor correlation between different commercial neprilysin RUO immunoassays is concerning and discordant findings need to be resolved. We seek to identify and validate reliable neprilysin immunoassays to strengthen the scientific rigor and reproducibility of neprilysin-related investigation and of biomarker research in general. METHODS: Soluble neprilysin (sNEP) concentrations were determined in cohorts (n = 532) from Spain (Cohort 1), New Zealand (NZ, Cohort 2) and Singapore (Cohort 3), using commercial kits from six vendors. Apparent sNEP concentrations were correlated between different assays and with plasma neprilysin activity. Assay reliability was further validated by performance verification, MS analysis and cross-reactivity tests. RESULTS: sNEP in Cohorts 1 and 2 measured concurrently in Spain and NZ showed significant inter-laboratory correlation only for the Aviscera Bioscience sNEP ELISA SK00724-01. Neprilysin concentrations obtained with the R&D systems and SK00724-01 ELISAs correlated with each other but not with neprilysin activity. In Cohort 3, sNEP concentrations from the Perkin Elmer AlphaLISA and Biotechne ELLA assays agreed (r = 0.89) and both correlated with neprilysin activity (r = 0.87, 0.77 respectively). MS analysis detected authentic neprilysin in the AlphaLISA kit calibrator and in antibody pull-down material from human plasma. The AlphaLISA assay performed within acceptable limits (spike and recovery, dilutional linearity, inter- and intra-assay CV) and showed no cross-reactivity against neprilysin substrates and closely-related analogues. CONCLUSION: AlphaLISA and ELLA assays provide reliable measures of sNEP concentrations. Reliability of other commercial neprilysin assays remains in question.

Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation.

High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13-24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets.

Ankyrin Repeat Domain 1 Protein: A Functionally Pleiotropic Protein with Cardiac Biomarker Potential.

The ankyrin repeat domain 1 (ANKRD1) protein is a cardiac-specific stress-response protein that is part of the muscle ankyrin repeat protein family. ANKRD1 is functionally pleiotropic, playing pivotal roles in transcriptional regulation, sarcomere assembly and mechano-sensing in the heart. Importantly, cardiac ANKRD1 has been shown to be highly induced in various cardiomyopathies and in heart failure, although it is still unclear what impact this may have on the pathophysiology of heart failure. This review aims at highlighting the known properties, functions and regulation of ANKRD1, with focus on the underlying mechanisms that may be involved. The current views on the actions of ANKRD1 in cardiovascular disease and its utility as a candidate cardiac biomarker with diagnostic and/or prognostic potential are also discussed. More studies of ANKRD1 are warranted to obtain deeper functional insights into this molecule to allow assessment of its potential clinical applications as a diagnostic or prognostic marker and/or as a possible therapeutic target.