Article ; Online: Finding a reliable assay for soluble neprilysin.
2022 Volume 104, Page(s) 51–58
Abstract: 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 ... ...
Abstract | 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. |
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MeSH term(s) | Biomarkers ; Enzyme-Linked Immunosorbent Assay ; Heart Failure ; Humans ; Neprilysin ; Reproducibility of Results ; Spain |
Chemical Substances | Biomarkers ; Neprilysin (EC 3.4.24.11) |
Language | English |
Publishing date | 2022-03-21 |
Publishing country | United States |
Document type | Journal Article |
ZDB-ID | 390372-2 |
ISSN | 1873-2933 ; 0009-9120 |
ISSN (online) | 1873-2933 |
ISSN | 0009-9120 |
DOI | 10.1016/j.clinbiochem.2022.03.004 |
Database | MEDical Literature Analysis and Retrieval System OnLINE |
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